A structural approach towards cell adhesion modulation and protein engineering

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DIPARTIMENTO DI

CHIMICA, MATERIALI E INGEGNERIA CHIMICA “Giulio Natta”

Dottorato di Ricerca in Chimica Industriale e Ingegneria Chimica (CII)

PhD in Industrial Chemistry and Chemical Engineering

XXIX ciclo 2014 - 2016

A STRUCTURAL APPROACH

TOWARDS CELL ADHESION

MODULATION AND PROTEIN

ENGINEERING

Matricola 814045

Coordinatore: prof. Alessio Frassoldati

Tutore: prof. Roberto Piazza

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5. Structural characterization and engineering of the deglycating enzyme

Amadoriase I...80

5.1. Crystal structure of free and substrate-bound Amadoriase I...81

6. Experimental procedures...87

6.1. Heterologous protein expression...88

6.2. Fundaments of chromatography...90

6.2.1. Affinity chromatography...91

6.2.2. Size-exclusion chromatography...92

6.2.3. Ion exchange chromatography...93

6.3. Crystallization techniques...93

6.4. X-ray diffraction...97

6.4.1. Crystal structure solution and refinement...100

6.4.1.1. Direct methods...101

6.4.1.2. Macromolecule structure solution methods...101

6.4.2. X-ray sources...104

6.5. Materials and methods...106

6.5.1. Gene cloning and protein expression...106

6.5.1.1. Cloning...106

6.5.1.2. Protein expression and purification...110

6.5.1.3. Protein crystallization...112

6.5.1.4. Crystallographic tables...114

6.5.2. Mammalian cell culture and cell-aggregation assay...116

6.5.3. Chemical immobilization of E-cadherin EC1-EC2...116

6.5.3.1. Substrate activation and silanization...116

6.5.3.2. APTES functionalization, first test...116

6.5.3.3. Functionalization during QCM experiment...117

6.5.3.4. Final functionalization condition used...117

Concluding remarks...119

Bibliography...122

Appendix I...133

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Index of figures and tables:

Fig. 1.1: schematic depiction of the organization of the cadherin extracellular domains. By convention, the numbering of the ECs is from the outermost (EC1) to the membrane proximal domain (EC5). Pag. 4.

Fig. 1.2: schematic depiction of the proteins involved in adherens junctions formation. Cadherins (in green) bind to other cadherins on the partner cell with their extracellular portion while with their cytoplasmic tail they interact with p120 catenin (orange) and β-catenin (red). This last protein forms a complex with α-β-catenin (blue), which in turns binds to F-actin (pink). This chain of molecular interactions allows for a mechanical link to be formed between the different cells of a tissue. Pag. 5.

Fig. 1.3: pictorial representation of two attached polarized epithelial cells, displaying the location of the different junctions and the related cytoskeleton portions. Adapted from16_.

Pag. 8.

Fig. 1.4: (A) electron microscopy image of a desmosome. (B) Schematic arrangement of the proteins that form the desmosomes. Desmocollin and desmoglein perform Ca2+-dependent cell-cell adhesion via their ectodomain while the C-terminus is placed, on the other side of the plasma membrane, into the outer dense plaque (ODP). Here, the linker proteins plakoglobin (PG), plakophillin (PKP) and desmoplakin (DP) form a complex that connect cadherins with the intermediate filaments (IF). The measurement reported are the distance from the plasma membrane. From21_{. Pag. 11.}

Fig. 1.5: cartoon representation of a cadherin EC (left) and its topology diagram. Adapted from31_{. Pag. 13.}

Fig. 1.6: phylogenetic tree of the cadherin superfamily. The cadherin major branch is depicted at the top and the cadherin-related major branch at the bottom. Some subfamilies are grouped in neither of the two major branches. while some cadherin-like proteins have not been completely classified yet. Figure adapted from34_{. Pag. 16.}

Fig. 1.7: schematic representation of the three main conformations that may adopt cadherins. (A) monomeric, (B) x-dimer and (C) strand swap dimer. Pag. 17.

Fig. 1.8: sequence alignment of the EC1-EC2 portion of different members of the classical cadherin type I sub-family, including the sequences of the same cadherin of different organisms. Clearly, some residues or short sequences, like Trp(W)2 or

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His(H)79-IV

Ala(A)80-Val(V)81, are well conserved throughout the entire family and all the species. Pag. 19.

Fig. 1.9: cartoon representation of the binding mode of classical type I cadherins as found in the structure of mouse E-cadherin EC1 (PDB code: 1NCI), with one monomer in green and the adhesion arm of the partner molecule depicted in purple. (A) Stacking arrangement of the Trp2 between Glu89 and Ile92 with the H-bond with the main chain of Asn90. (B) hydrophobic residues lining the pocket that hosts the Trp2 (not shown). Pag. 20.

Fig. 1.10: (A) detail of the calcium binding region and (B) cartoon representation of the x-dimer conformation (PDB code: 1EDH). Pag. 22.

Fig. 1.11: ribbon representation of the architecture of the full extracellular portion of mouse C-cadherin (PDB code: 1L3W). The circles highlight the three calcium ions, represented as green spheres, located in each linker region between the ECs. Adapted from 196_{. Pag. 23.}

Fig. 1.12: (A) 90° rotated cartoon representation of the strand swap dimer found in the crystal structure of the full ectodomain of C-cadherin (1L3W, green) and (B) parallel packing of the ectodomains showing the cis interaction. (C) Superimposition of the strand swapped dimers formed by the EC1-EC5 fragment of C-cadherin, E-cadherin (3Q2V, cyan) and N-cadherin (3Q2W, pink). Pag. 25.

Fig. 1.13: cartoon representation of the arm conformation found in the x-dimer (A) and in the two structures of human P-cadherin showing both x-dimer and strand swap (B-C). The conformation shown in figure B presents a curved adhesion arm with the pocket filled by Met0 and Trp2 docked in a second hydrophobic cavity below of it. Conversely, in the last conformation (C) the adhesion arm is more relaxed, with the Trp2 fitted in the adhesion pocket despite the extra residue at the N-terminus. PDB codes: 1FF5, 4ZMT and 4ZMV respectively. Pag. 27.

Fig. 1.14: current model of the cadherin dimerization pathway. The conformations that are depicted in blue in the diagram are still not confirmed experimentally. In solution, the monomer is in equilibrium between a closed and an open state (A and A' respectively). Then, it can either undergo a slow direct dimerization process (direct arrows to D) or form a series of intermediate states (x-dimer) that lower the energy associated with the arm opening process that leads to strand swap dimer formation. After x-dimer formation (B) the arm opens and slides the Trp2 residue toward the partner pocket (C and C') along the EC1, while in the meantime varying the angle between the 2 units and finally forming the strand swapped dimer (D). The states

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depicted in blue (C and C'), although representing reasonable "snapshots" of the evolution of the system from the x-dimer, still need experimental validation. Pag. 28. Fig. 1.15: artistic depiction of protein glycation reaction pathway. Circulating glucose (1) reacts with a free amine (a lysine side-chain of collagen, in the picture) (2) producing a Schiff base (3) which spontaneously rearranges into the Amadori product (4). Over time, the Amadori product undergoes different reactions and rearrangements (in the picture the crosslinking with an arginine) that lead to several different AGEs (5). Pag. 32.

Fig. 1.16: structures of glucosepane (left) and its precursor dideoxysone (right). Pag. 33. Fig. 1.17: reaction mechanism of a FAOX enzyme. The C-N bond between the amino-acid moiety and the sugar moiety of the glycated molecule is first oxidized, thanks to the FAD, to an imine (1) that is then hydrolyzed, yielding glucosone and the free amino-acid amine. From125_{. Pag. 35.}

Fig. 2.1: SDS-PAGE of P-cadherin EC1-EC2 (A-B) and P-cadherin EC1-EC4 (C-D) taken at different purification steps. In the pictures, the spots corresponding to the eluted fractions are also highlighted. From the gels after Ni-affinity chromatography (A and C) it is evident that both the proteins are expressed in very large quantity by the bacteria but that this step is not sufficient to have a completely pure product. After a subsequent the gel filtration step (B and D), both protein fragments are obtained at a high purity grade. Pag. 40.

Fig. 2.2: optical microscope image of crystals of human P-cadherin EC1-EC2 (left, 150x50x50 µm) and EC1-EC4 (right, 150x150x30 µm). Pag. 41.

Fig. 2.3: (A) structure of human P-cadherin EC1-EC2, showing a monomeric and antiparallel packing arrangement; (B) detail of the water-mediated interactions between the adhesion arm and the BC-loop of the second domain of a symmetry related molecule; (C) sequence alignment showing the BC-loop of domain 2 of different type I classical cadherins. Pag. 42.

Fig. 2.4: (A-D) panels showing the attribution process of the two different conformation of Trp2 during the model refinement phase. In the two intermediate panels (B and C) is clearly visible how, by placing the tryptophan in only one of the two possible conformations, some unassigned electron density remains beside its side chain (the green blob), which then disappears once both the conformations are fitted in, with a 50% occupancy each. (E) Cartoon representation of the final human P-cadherin EC1-EC2 model showing, as sticks, the Trp2 and the residues involving in its binding inside the pocket. Pag. 44.

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Fig. 2.5: (A) free energy surfaces along s(R) and z(R), for E- (top) and P-cadherin (bottom). Gaussians deposited every 1 ps with a width of 0.1, and 0.5 Å, along s(R), and z(R), respectively. Both systems were simulated for 500 ns within the canonical NVT ensemble, at 300K. (B) The free energy profiles optimized on the LFEP by means of US simulations. These profiles are associated to a transition between basin I and basin II, occurring in E- (black solid line), and P-cadherin (black dotted line). Pag.46.

Fig. 2.6: crystals obtained for human P-cadherin EC1-EC2 Ala5Pro (left, 200x30x30 µm) and Val4Ile-Ala5Pro (right, 100x20x20 µm). Pag. 48.

Fig. 2.7: schematic depiction of the different angles found between different cadherins EC1-EC2 fragments. As it can be seen, compared with the wild type, human P-cadherin EC1-EC2 mutants has a very different angle. Pag. 49.

Fig. 2.8: cartoon visualization of the two different types of interaction observed in the crystal structure of the two mutants of human P-cadherin EC1-EC2 (A); the strand swap dimer is formed by the green and the purple molecules, while the purple and the yellow are coiled in an anti-parallel stack. Below (B), the magnified detail of the interface involving the HAV (residues 79-81) and PGT (residues 123-125) triplets from the two interacting molecules. Pag. 50.

Fig. 3.1: structure of ADH-1 and of the compounds we developed and, on the right, a graph showing the cadherin adhesion inhibition. Pag. 55.

Fig. 3.2: cartoon representation of the area of the x-dimer between the EC1s of the two interacting cadherins. In the structure of the sole protein (A) this area is occupied by water molecules, while in the structure of the complex (B) a long electron density "blob" corresponding to the inhibitor is clearly visible. Pag. 57.

Fig. 3.3: stabilizing contacts between the ligand and the protein. As mentioned in the text, for simplicity the ligand is shown in only one of the two possible orientations. Due to the nature of FR159 and of the residues involved, the contact are mostly hydrophobic, with the exception of a H-bond between one carbonyl of the heterocycle, the one opposed to the phenyl ring, and the amide nitrogen of Ser8. Pag. 58.

Fig. 3.4: microscope visualization of the stable knockdown of E- and P-cadherin. Cadherin expression was assessed by immunostaining in BxPC3 shRNA cells. Cells were fixed and stained with anti-E-cadherin or anti-P-cadherin Abs. After washing, cells were then incubated with Alexa-488 conjugated goat anti-mouse or Alexa-488 conjugated goat

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anti-rabbit Abs and were observed with a confocal microscope. Cell nuclei were stained with Draq5. Scale bar: 100 µm. Pag. 63.

Fig. 3.5: screen for DMSO tolerance for BxPC-3 sh-ctrl cells. Spheroids formed after 24h at different DMSO concentrations. At 2.5% DMSO concentration cell viability limit could be identified based on these preliminary tests. Cell viability does in fact appear to be completely compromised starting at 5% DMSO concentration. Pag. 64.

Fig. 3.6: cell viability rate of BxPC3 sh-CTRL, sh-Ecadh and sh-Pcadh in increasing concentration of DMSO: Cells were treated with various concentrations of DMSO for 24h or 48h and then incubated with MTT for 3h. Cells viability was quantified by measuring the optical density at 570nm. The graph represents the mean of 2 independent experiments. The significance is calculated with a Mann and Whitney test (p-value < 0,001 = ***). Each condition is compared to the 0% DMSO condition. Pag. 64.

Fig. 3.7: screen for DMSO tolerance for BxPC-3 sh-E and sh-P cells. Spheroids formed after 24h at different DMSO concentrations. At the 3% DMSO concentration, cell viability starts to be compromised in both cases, while at 2% they still have a similar appearance to those at lower DMSO concentrations. Pag. 65.

Fig. 3.8: (top) images of the spheroid formed by BxPC-3 sh-ctrl cells in 2% DMSO and of the results of the addiction of the inhibitors. Only those molecules that showed inhibitory activity are reported. In some well some precipitate is clearly visible while AS09 is marked with an asterisk since the precipitate was removed by centrifugation before the seeding of the cells; for this sample the actual concentration of the inhibitor is unknown. (bottom) Relative area (%) of the spheroids grown in the presence of 1 mM modulators normalized on that of those grown with just 2% DMSO. AS08, AS09, AS19, LC02 and LC11 are not shown since the spheroids were too extended or not formed at all. Pag. 66.

Fig. 3.9: images of the spheroid formed by BxPC-3 sh-ctrl cells added with 0.1 mM inhibitor. As previously done, a sample with just 2% DMSO and no inhibitor was used as a control. Relative area (%) of the spheroids grown in presence of 0.1 mM modulators normalized on that of those grown with just 2% DMSO. Again AS09 is not reported since no formation of a spheroid could be observed. Pag. 67.

Fig. 3.10: screen with AS09 at 0.05 mM concentration and AS11 at 0.1 mM concentration with 2% DMSO on the three BxPC-3 cell lines. Screen of the 2 best inhibitors, AS09 and AS11, with the cell lines expressing just E- or P-cadherin. Notably, at the 0.05 mM concentration AS09 only works on E-cadherin while the activity of AS11 on P-cadherin can be appreciated by the morphology of the spheroid rather than by its area. Pag. 68.

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Fig. 4.1: picture of the SDS-PAGE gel after the first affinity purification step. Clearly, two expression bands are present, one corresponding to the monomer and the other to the dimer generated by the formation of a disulfide bond between two C-terminal cysteines. Pag. 73.

Fig. 4.2: schematic representation of the two different behavior of the protein prior and upon cleavage of the histidine-tag. With the tag on (top), the protein is inactive and therefore, upon oxidation and disulfide bond formation, it forms a dimer that is still soluble. When the histidine-tag is removed and the protein is activated (bottom), dimerization through disulfide bond formation combines with the natural cadherin dimerization mechanism, causing the formation of long chains and protein precipitation. Pag. 74.

Fig. 4.3: SDS-PAGE gel after the first affinity purification step (A) and after the enzymatic cleavage of the histidine-tag (B) and therefore protein activation. Clearly, the band corresponding to the dimer is very small and almost all the protein is present as monomer both in the inactive and in the active state. Pag. 75.

Fig. 4.4: fluorescence microscope image of untreated glass dipped in protein-FITC solution (A) and of the sample (glass) functionalized according the GOPS pathway, the protein identified by reaction with FITC. Both pictures were taken after SDS wash. Scalebar 50 µm. Pag. 77.

Fig. 4.5: APTES-glass coupled only with 6-maleimidohexanoic acid (A-C); brightfield, fluorescence and superimposition, respectively. Glass fully functionalized according to the APTES route (D-F); brightfield, fluorescence and superimposition, respectively; Both samples were treated with 1 mM FITC in PBS pH 8.0 for 7h prior to visualization. Scalebar 50 µm. Pag. 78.

Fig. 5.1: superimposition of the crystal structure of Amadoriase I (green, PDB code: 4WCT) and inhibited Amadoriase II (purple, PDB code: 3DJE). The red circle indicates the two loops that were found to be in different conformations in the two structures. Pag. 82.

Fig. 5.2: omit map of the electron density of the ε-fructosyl-lysine in the cavity of the enzyme shown at three different occupancy values of the substrate (0.1, 0.3 and 0.5, from left to right). It can be clearly seen that the unassigned electron density decreases as the occupancy value increase. Pag. 84.

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Fig. 5.3: (A) cartoon representation of the crystal structure of the inhibited form of Amadoriase I (PDB code: 4XWZ) and (B) stabilizing contacts formed by the substrate in the catalytic cavity. The interacting residues are represented as sticks. At the center of the image is also shown the water molecule that putatively hydrolyzes the imine formed after the oxidation of the C-N bond by the FAD, splitting the substrate in two. Pag. 84. Fig. 6.1: schematics of the chromatography technique. The mixture is loaded on top of the stationary phase and pushed through it with the eluent. With time, since the different components interact with different strength to the stationary phase, they gradually separate until they are eluted separately from the column. Pag. 91.

Fig. 6.2: schematic depiction of the functioning of a metal-affinity column. (A) At the beginning the metal loaded resin (Ni2+ in the example) the coordination of the ion is completed by water molecules. (B) Upon flowing the protein onto the resin, the imidazole moieties of the histidine-tag displace the waters from the nickel coordination and, as a consequence, they bind the protein to the resin. (C) To elute the protein, the resin is then treated with a solution containing imidazole which, due to its higher concentration, displaces the protein allowing us to recover it. Pag. 92.

Fig. 6.3: phase diagram for protein crystallization. The solid line separates the under-saturated region from the superunder-saturated ones and corresponds to the maximum protein solubility at the specific precipitant concentration, while the separations between the three supersaturated areas are not so well defined. From197_{. Pag. 94.}

Fig. 6.4: schematic depiction of the experimental setup for hanging drop (left) and sitting drop (right). Pag. 96.

Fig. 6.5: schematic depiction of Bragg's law (left) and the Ewald sphere (right). Pag. 99. Fig. 6.6: histogram plot of the number of deposited structures (left) and number of overall folds (right) from 1990 to nowadays. Data from www.rcsb.org. Pag. 102.

Fig. 6.7: pictorial representation of the molecular replacement procedure. The model molecule, in green, is fitted to the target protein, with a combination of rotations (R) and translations (T). Pag. 103.

Table 6.1: thermocycler program. Pag. 109.

Table 6.2: minimal medium composition. Pag. 110. Table 6.3: SDS-PAGE gel preparation. Pag. 112.

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Table 6.4: protein crystallization conditions. Pag. 113.

Table 6.5: crystallographic table for the structures of wild type and mutated human P-cadherin EC1-EC2 (chapter 2). Pag. 114.

Table 6.6: crystallographic table for the structures of E-cadherin (Val3) EV1-EC2 free and in complex with FR159 (chapter 3). Pag. 115.

Table 6.7: crystallographic table of the structures for the structures of free and substrate-bound Amadoriase I (chapter 5). Pag. 115.

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Abstract

Protein X-ray crystallography is a very powerful tool that provides, at the molecular level, the high quality and high resolution structural information that is necessary for structure-function correlation analysis, protein engineering, structure based drug design and molecular dynamics (MD) simulations.

In this thesis, this technique has been extensively used for the investigation of the structure, properties and possible application of the extracellular portion of some selected cell-adhesion proteins of the type I classical cadherin family and of a deglycating enzyme from the large Fructosyl Amino Oxidase family, called Amadoriase I.

Cadherins are calcium-dependent trans-membrane proteins that comprise three clearly discernible regions: intracellular, trans-membrane and extracellular. The extracellular portion is formed by a variable number of so-called extracellular cadherin (EC) repeat domains, each formed by approximately 110 amino acidic residues and rigidified by the presence of Ca2+_{ions between them.}

They are of critical importance for cell-cell adhesion, a fundamental process that results in cellular organization and tissue differentiation, thus allowing the formation of tissues and organs and ultimately the development of complex multicellular organisms.

Other than their adhesive function, cadherins also perform a cell-cell signaling function. It is well known that variations in cadherin natural expression level and changes in their ability to form either homophilic dimers or bind to selected protein substrates correlate with the onset and the progression of diseases such as cancer, asthma and chronic inflammation states.

Although the cadherin main functions (adhesion and signaling) have been quite extensively investigated over the last two decades, the mechanism by which such tasks are performed still needs to be fully elucidated. Over time, a combination of biophysical techniques (mainly X-ray diffraction, NMR and SAXS) have provided a clear, albeit still incomplete, picture of the adhesion mechanism, leaving the complete trajectory that leads, very dynamically, from the monomer to the dimeric adhesive state and back still partially elusive. The implications of such lack of complete structural characterization are important: to date, no clear molecular bases for cadherin homo-selectivity and for their energy activation profiles have been unambiguously identified.

In this thesis work, I have studied selected cadherin family members at the molecular level by single crystal X-ray crystallography. Overall, my goal was to contribute to the characterization of their adhesion mechanism. Furthermore, in the context of this thesis, I combined this structural analysis with biophysical, computational and functional studies aimed at the development or the identification of molecules that are capable of modulating cadherin homophilic adhesion. Finally, starting from the structural information, I have engineered a cadherin family member with the goal of producing

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functionalized biomaterials. In fact, due to the important role of the protein in tissue sorting and formation, such cadherin-functionalized hybrid materials may be employed as scaffolds in tissue engineering and tissue regeneration or be used for the development of cell-sorting or sensing lab-on-chip platforms.

The second project that I focused on in my thesis is the X-ray crystal structure of the deglycating enzyme Amadoriase I. A member of the Fructosyl Amino Oxidase family, this enzyme is capable of hydrolyzing the bond between the amine and the sugar moiety of a glycated amino acid.

From a biomedical point of view, the study of this enzyme is very important since protein glycation reactions occur spontaneously in the body over time due to the sugar present in blood. As a result of this spontaneous glycation reaction (usually referred to as the Maillard reaction) proteins are progressively glycated and, especially those with long half-life, tend to become heavily crosslinked over time. These non specific modifications negatively affect the function of the proteins and may eventually lead to the development of diseases such as Alzheimer’s disease, arteriosclerosis, nephropathy and retinopathy, with a higher incidence in elderly people and in people with abnormally high blood sugar levels.

Moreover, deglycating enzymes are also utilized for the measurement of the concentration of the glycated form of hemoglobin (HbA1c) in the blood. In fact, due to the relatively long life-time of hemoglobin (90-120 days) and therefore its tendency to be glycated over time, this is a very good indicator of the concentration of blood glucose over a period of 2-3 months.

Unfortunately, however, due to the fact that the available enzymes only work on glycated amino acids, this process is not immediate since HbA1c must be proteolytically cleaved before the enzymatic detection of selected glycated amino acids can actually take place.

During my thesis, I contributed to the structural characterization of of the apo form and the substrate-bound form of Amadoriase I from Aspergillus fumigatus. Moreover, I have been actively involved in the first attempts to engineer this enzyme in order to enhance its natural substrate recognition capabilities. All these studies have been conducted using a combination of molecular biology, protein chemistry, X-ray crystallography and molecular dynamics techniques. The ultimate goal with this project is to engineer the Amadoriase I enzyme in order to enlarge its catalytic cavity and allow its catalytic activity on large substrates such as polypeptides or even whole glycated proteins. This would, for instance, potentially improve the current procedures employed for the measurement of HbA1c in diabetic patients, possibly leading to new, efficient and low cost diagnostic tools for diabetes monitoring. Other potential applications of such molecular technology include the use of such chimeric enzyme to slow or even reverse collagen rigidification in aging tissues as well as in the food industry, to limit protein glycation in aliments that need thermal treatment (e.g. UHT milk).

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For the experimental work described in this thesis, X-ray crystallography is used as main investigation tool, providing not only base information on the structure-function relationship of the investigated proteins, but also constituting the starting point to perform drug-discovery and protein engineering.

Although the instruments and techniques used to carry on the different projects that will be described herein are mostly the same, the research work has been performed onto two very different sistems. In fact, while the majority of the research dealt with some selected members of cadherin cell-cell adhesion protein family (chapters 2 to 4), exploring the structural basis of their different activity, their possible use in the developement of bio-materials and the discovery of small-molecule modulators, an important portion of the work, carried out in close collaboration with the group of Prof. Simone Vesentini at the Politecnico di Milano, dealt with the structural characterization and engineering of a deglycating enzyme of the amadoriase family (chapter 5).

In this chapter the basis for the comprehension of the experimental work will be provided along with a general overview of the state of the art about the two system treated and each chapter presenting the experimental results will also provide a further framing of the discussed topic.

1.1. Cell-cell adhesion

In the course of evolution, a crucial step toward the development of complex organisms has been the passage from unicellular to multicellular living systems. This advancement required the differentiation of cells, their specialization in one or several precise functions and their selective aggregation to form tissues, where cells are connected and communicate to each other at the mechanical, electrical and chemical level. Tissues finally associate in various combinations, originating specialized organs, where a tight cooperation between cells takes place to perform complex functions. In humans, hundreds of different types of specialized cells are grouped in just five classes of tissues: epithelial, nerve, muscle, connective and lymphoid.

A key feature of this increase in complexity is the possibility for the cells to be physically connected to each other, a task that can be achieved in two different ways: cells may indirectly bind to each other by interacting, through adhesion receptors on their membrane, with proteins and polysaccharides secreted in the intercellular space (the extracellular matrix, ECM) or they can directly bind to each other through specialized integral proteins generally referred to as cell adhesion molecules (CAMs). Most of the CAMs can be grouped in four major superfamilies: integrins, selectins, immunoglobulins and cadherins. Depending on their nature, these molecules can mediate the adhesive interaction between cells of the same type or of different type (homotypic and heterotypic binding, respectively) while they can perform their function by binding to

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the same CAM of the surface of adjacent cell (homophilic binding) or to a different one, even from a different superfamily (heterophilic binding).

Cell-cell adhesion and cell-matrix adhesion molecules are therefore ultimately responsible for the formation, the function and the architecture of tissues. Hence, the evolution of multicellular organisms, with complex tissues and organs, directly depends on the divergent evolution of these molecules and on their differentiation.

Of fundamental importance for the evolution of complex multicellular organism is the epithelial tissue; the arrangement of the cells in the epithelial sheet has, in prospective, the same evolutionary significance than that of the cell membrane for complex single cells.1

The epithelial tissue lines almost every free surface of the body, constituting not only a boundary between the different compartment of the body but also, thanks to specialized junctions between the cells, a barrier towards water, solutes, other cells and, in general, the external environment.

As for every process in living organisms, also cell-cell adhesion is performed and regulated by a complex and diverse ensemble of molecules that have long been the subject of extensive research by the scientific community.

In the 1970s, the first investigations on membrane proteins mediating cell-cell adhesion resulted in the identification of a calcium-dependent glycoprotein with a molecular weight of 84 KDa involved in morula compaction and blastocysts formation named gp84 (from glycoprotein of 84 KDa). Since antibodies targeting this molecule turned the morula into a cell aggregate resembling grapes (uva in Latin), the protein was later renamed uvomorulin. Finally, in 1984 it became clear that uvomorulin is a member of a large class of calcium-dependent cell adhesion proteins and the acronym cadherins was introduced.

The first proteins of this family to be thoroughly investigated were those found in the adherens junction (AJ) of epithelial cells. These proteins are nowadays referred to as classical cadherins and they are the most studied among all members of the large cadherin superfamily.2,3

Today, the different members of the classical cadherins subfamily are conventionally named after the tissue in which they were first discovered and marked with its initial. For instance E-cadherin for epithelial, N-cadherin for neuronal, R-cadherin for retinal, P-cadherin for placental, etc.

Cadherins perform their adhesive function through their extracellular portion, which comprises a variable number of immunoglobulin-like domains arranged in tandem and connected by short linker regions. Three calcium ions are found in close proximity to each linker region, at specific binding sites. These Ig-like domains are usually referred to as cadherin extracellular (EC) domains. In all classical cadherins, the whole extracellular portion is composed of five ECs (EC1-EC5, where EC5 is the membrane-proximal domain).

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Generally speaking, a pair of cadherin protruding from opposing cells interact through their extracellular portion forming a dimer using their two most external domains (EC1 and EC2). Although the affinity in this interaction is not particularly high (incidentally, this is a feature shared by a significant portion of the plasma receptors) and although all the different cadherins show a high sequence homology, cadherin association is very specific as it almost exclusively occurs between identical members of the family.4

This low affinity characteristic is related to the function of the protein. Indeed, other than their mere adhesive function, cadherins are also involved in conferring stability, resistance and other mechanical properties to the tissue, not only favoring its role as a physical barrier but also allowing the continuous rearrangement of the cells and of the extracellular matrix, hence their reciprocal interactions. Moreover, despite the relative weakness of the single interaction, a large number of interactions participate simultaneously in the adhesion process, thus ensuring the stability of the adherens junction. The whole cellular adhesion process is therefore driven not just by affinity but also by avidity, a feature that is also referred to as "Velcro effect".5

Other than the dynamics associated with the reversible formation of the individual dimers, at the cell-cell junction a further equilibrium exists between the molecules that are already present in-site and those that freely diffuse in and from the cytoplasm, a process regulated by a trafficking mechanism.6

After being synthesized, cadherins are moved from the Golgi apparatus to the surface of the cell; at this stage, a cadherin also usually binds β-catenin, an armadillo protein that mediates the interaction between the cytoplasmic domain of classical cadherins and the actin filaments. It is worth stressing that the cadherin cytoplasmic tail also crucially contributes to sorting signals.

Cadherin expression levels are not constant. Rather, they depend on the status of the cell: for epithelial cells, for instance, there is a great production and trafficking of

E-Fig. 1.1: schematic depiction of the organization of the cadherin extracellular domains. By

convention, the numbering of the ECs is from the outermost (EC1) to the membrane proximal domain (EC5).

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cadherin in non-adherent and pre-confluent cells, whereas in the mature epithelium their quantity is much lower since the cadherin-mediated junctions are already formed and positioned.6

E-cadherin allows for the correct trafficking in the basolateral membrane and, with its positioning on the cell membrane, allows for the correct polarization of epithelial cells. Furthermore, by interacting with the Sec6/8 complex, a protein complex that lines the areas for vesicle fusion site in the apicolateral area of the plasma membrane, de facto orchestrates its correct localization.7

Finally, cadherins mediate the initial contact between cells and, after being correctly placed and organized on the plasma membrane, allows for the positioning of the other integral proteins that participate in the cell-cell adhesion process.

Since cadherins connect the interior with the exterior of the cell, upon dimerization they also trigger a rearrangement of the actin cytoskeleton to which they are connected through cytoplasmic armadillo proteins that belong to the catenin family. Beside β-catenin, α-catenin and p120 catenin also participate in the binding.8

These proteins bind the intracellular portion of cadherins from the beginning of their lifetime and it has in fact been demonstrated that the cytoplasmic domain of cadherins is unfolded in the absence of β-catenin.9

Since the cadherin-β-catenin binding site also contains a sequence recognized by ubiquitin, the formation and the disruption of such complex also regulates the turnover

Fig. 1.2: schematic depiction of the proteins involved in adherens junctions formation. Cadherins (in

green) bind to other cadherins on the partner cell with their extracellular portion while with their cytoplasmic tail they interact with p120 catenin (orange) and β-catenin (red). This last protein forms a complex with α-catenin (blue), which in turns binds to F-actin (pink). This chain of molecular interactions allows for a mechanical link to be formed between the different cells of a tissue.

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of the protein. β-catenin binds the cytoplasmic end of cadherin through its 12 armadillo repeats at a site located at the N-terminus of α-catenin, although both ends of the protein appear disordered in the crystal structure.10

Although clearly of utmost importance, the function and the interaction network of α-catenin is still quite debated; in fact, very few putative partners other than itself and β-catenin have univocally been identified to date.11

The homo-dimerization equilibrium does not only compete with the hetero-dimerization process in terms of number of molecules that are available, but also physically, since the areas that are involved in these two distinct complexes partially overlap, the α/β being bigger and thus accounting for the observed preference for heterodimer formation in solution.11

Another known α-catenin ligand is afadin, a protein to whom α-catenin binds through its M-domain. This protein consists of two domains formed by four helices each and connected by a linker. Afadin undergoes large conformational changes upon binding to α-catenin.12

Finally α-catenin binds through its C-terminus to F-actin, although the details of this interaction are still not clear. In fact, although at first a direct link between the cadherin/catenin complex and the actin filaments was believed to occur, this hypothesis was later confuted by the demonstration that such binding is inhibited by β-catenin. Nowadays, a more accredited hypothesis is that the recognition between the cadherin/catenin complex and the actin filaments occurs through transient interactions in which α-catenin adopts different conformations. However, this set of interactions and the attribution of the role of catenin in the reorganization of F-actin in AJs are still a matter of extensive research.13,14

Lastly, p120 catenin binds the cadherin cytoplasmic portion at the level of the juxtamembrane domain, where it acts as a regulator of both the cadherin turnover and, by interacting with small GTPases, of the actin cytoskeleton.

Clearly, the interactions with F-actin, along with its polymerization and bundle formation, are far from simple and involve a large number of proteins, some of which, for instance spectrin, EPLIN and formin-1,15,16_{do not necessarily interact with catenins}

and therefore this topic will not be further examined in the context of this work.

The cadherin-catenin-actin complex described above is not the only key factor in AJs formation. In fact, these cell-cell junctions also contain a thick network of different transmembrane calcium-independent proteins.5

Owing to their structure , the vast majority of these proteins are grouped in the immunoglobulin CAM superfamily (IgSF CAM). Nectins, CTX and L1 families are the most prominent members of IgSF CAMs, along with some CAMs that are simply identified according to their tissue localization. Once again, the topic is very broad and goes beyond the scope of this thesis. Therefore, here I will discuss only some selected proteins, which are of particular interest in this context because of either their cell adhesion role or their cellular localization.

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Nectins and nectin-like molecules (necl) are a family of integral proteins involved in cell-cell adhesion that are also implicated in immune modulation and evasion. They comprise an extracellular portion composed by 3 Ig-like domains, a single pass transmembrane region and a cytoplasmic tail and they perform both homophilic and heterophilic binding. Like cadherins, these proteins are found in all kinds of tissues, although different members of the family may be found on different cells with an overlapping distribution.17_{Nectins, but not necl proteins, bind to afadin through their}

C-terminal cytoplasmic portion, and through this to F-actin and α-catenin.

Because of the sharing of these last interactions, the nectin/afadin complex is closely related to the cadherin/catenin/actin one, as they are both crucial for the cell-cell adhesion process. Although the overall mechanism is not yet fully understood, nectins also seem to directly interact with cadherins. In fact, as cell-cell contacts are forming, nectins localize at the apical portion of the cells contributing to the recruitment of several CAMs, including cadherins. This represents an alternative afadin-dependent recruiting mechanism.18_{In more recent years, a third cell adhesion machinery involving nectins}

and independent from both afadin and cadherins has been described extensively. In particular, it has been found in the olfactory bulb, in the mammary gland and in commisural axons. The apparatus has been named nectin spots due to its appearance, dots or short lines, at the immunofluorescence or electron microscope. Small clusters of nectins seem to be involved in weak and transient cell-cell adhesion, but it is also conceivable that they might also play some role in developing and reorganizing tissues, in cooperation with integrins and membrane receptors.17

Another prominent member of the IgSF CAM superfamily is N-CAM, which is found to be expressed mainly, but not exclusively, on neuronal cells. N-CAM exists in more than 20 different isoforms, all resulting from alternative splicing patterns of the same gene. As with the previous family, they are integral proteins with a small cytoplasmic domain, a single transmembrane portion and an ectodomain comprising a repetition of Ig-like domains and fibronectin type III domains. N-CAMs dimerize in a homophilic manner, contributing to cell-cell adhesion, although this process can be hampered by the post-translational addition of multiple molecule of the negatively charged sialic acid. Because of this process, these molecules mainly act as modulators of calcium-independent cell-cell adhesion, regulating the strength of the contact during development and tissue regeneration.5

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As previously discussed, the molecules involved in cell-cell and cell-matrix adhesion tend to localize and cluster in specific areas to perform their function. Such areas are usually referred to as junctions. Although specialized junctions are present in all tissues at different points of cell-cell or cell-matrix contacts, in this section I will only focus on epithelial cells not only because they are of particular abundance and importance but also because most of the work described in this thesis refers to cell adhesion proteins that are expressed in epithelial cells.

Within a tissue, epithelial cells are polarized and organized in three different regions: the apical (at the top), facing toward the exterior of the tissue, the basal, opposite to the previous one and contacting the extracellular matrix, and the lateral regions, between them; these last two areas host the adhesive macromolecule, cell-ECM and cell-cell respectively, and are sometimes collectively referred to as the basolateral region. Multiple types of junctions can be found at the interface between different cells or between the cell and the extracellular matrix and they can be broadly grouped in three categories, according to their role: occluding junctions, which seal cells together preventing the leakage of molecules from one side to the other of the epithelial layer; anchoring junctions, which physically attach the cell with the neighboring cells or the ECM and, through adaptor proteins, with the cell cytoskeleton; communicating junctions, which regulate the traffic of chemical and electrical signals in between adhering cells.

Fig. 1.3: pictorial representation of two attached polarized epithelial cells, displaying the location of

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1.2.1. Occluding junctions

Occluding junctions, also referred to as tight junctions, are patches of adhesive proteins that line the border between the apical and basolateral region of polarized epithelial cells and perform a function of sealing and segregation. They are mainly constituted by claudins, occludins and tricellulins that, as they protrude from the plasma membrane of opposite cells, form a series of strands that can be clearly seen by electron microscopy.19

In primis, tight junctions constitute the border between apical and basolateral regions of

the cell, preventing the diffusion of proteins from one domain to the other of the plasma membrane and thus preserving cell polarity. Secondly, they seal the space between adherent cells, so water soluble molecules cannot cross from one side to the other of the epithelial sheet. However, permeability to small molecules may greatly vary between different types of epithelia and may also be transiently changed, breaching the junctional barrier, in performing paracellular transport. For instance, epithelial cells lining the lumen of the intestine may loosen their adhesion in order to facilitate the absorption of monosaccharides or amino acids. In this tract, in fact, these species reach a high enough concentration to be passively transported to the other side of the cell layer.5

1.2.2. Anchoring junctions

Anchoring junctions guarantee the mechanical integrity of the tissues by allowing connecting cells to act as a unique structural unit. In fact, these type of junctions are found to be more abundant in tissues that are subjected to high mechanical stress, such as heart muscle or skin epithelia.

Generally speaking, anchoring junctions are constituted by plaques of intracellular attachment proteins, which bind to actin or intermediate filaments, and by transmembrane linker proteins, which bind on the intracellular side with one or more intracellular attachment molecules and with the extracellular portion with the extracellular matrix or with other transmembrane linker proteins protruding from the opposing cells.

Owing to this range of diverse binding modes, these junctions may be further divided into desmosomes and hemidesmosomes, which intracellularly connect with the intermediate filaments, and adherens junctions, which connect with actin instead.

However, although anchoring junctions can be grouped and described in different ways, the molecular basis of their functioning are shared and relatively simple: the transmembrane proteins dimerize and bind adjacent cells together or connect cells with the extracellular matrix. At the same time, intracellular proteins connect them to the actin cytoskeleton or the intermediate filaments. This tandem binding finally tie together all the interacting cells and the underlying extracellular matrix, which becomes a unique object.5

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1.2.2.1. Adherens Junctions

Adherens junctions, in a similar way to tight junctions, form a belt encircling the whole cell. They are located in the lateral domain of the plasma membrane, near the apical domain, just below the tight junctions. At the electron microscope, they appear as a dense plaque associated with a network of fibers belonging to the cytoskeleton.

The protein complex forming the adherens junction can be split in two areas: one extracellular, where adhesive proteins bind to their partner and hold cells together and one intracellular, where adaptor proteins connect the adhesive proteins with the cytoskeleton, thus forming a continuous object.

The bound cytoskeleton, which runs parallel to the plasma membrane, is constituted by polymerized units of actin, a globular protein, which in this form is referred to as F-actin or actin filaments.16,20

It is worth stressing that not only do these filaments have a strictly structural function, but their synthesis through actin polymerization and the formation of a complex with myosin, a motor protein, are at the basis of cell movement and folding of cell sheets into tubes and other structures.

From the F-actin, a bundle of actuator proteins, catenin, vinculin, α-actinin and plakoglobin, interact in different ways to connect the actin filaments with the integral proteins, cadherins and integrins, which, by dimerizing with partners on neighboring cells, complete cell-cell adhesion.

Both these transmembrane proteins depend on calcium to work properly, as can be inferred by the detachment of cultured cell monolayers upon treatment with calcium-chelating agents such as EGTA. This, however, is not enough to completely abolish cell-cell adhesion since, as discussed, calcium-independent adhesion mechanisms are also available.

1.2.2.2. Desmosomes

Differently from tight and adherens junctions, desmosomes are spot-like junctions. They are located on the lateral portion of the plasma membrane. Desmosomes shape the cell and help distributing shear forces by connecting the intermediate filaments of one cell with those of neighboring ones.

Intermediate filaments are components of the cytoskeleton. They are formed by subunits, which are in turn constituted by macromolecules that may differ among different tissues (for instance they are made of vimentin in the mesenchyme and keratin in the epithelium).

Desmosomes comprise three different areas: the extracellular region, the outer dense plaque and the inner dense plaque. Like other junctional complexes, they are formed by many different components, which mainly belong to the cadherin, plakins and armadillo protein superfamilies.21

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Cadherins found in desmosomes are classified as desmocollins and desmogleins. As with other cadherin family members, they provide calcium-dependent cell-cell adhesion through their ectodomains, which are composed by five Ig-like repeats.22_{Plakins that are}

found in desmosomes are usually referred to as desmoplakins and they are the most abundant proteins in such structures. In their active state, these macromolecules are found as homodimers and they bind through their carboxylic end to the intermediate filaments. Finally, the armadillo proteins plakoglobin and plakophilin complete the chain mediating the connection between cadherins and desmoplakins.23

The role of desmosomes is critical for tissues integrity. Indeed, multiple diseases are known to compromise their function. One of the most known condition is the pemphigus, an auto-immune disease characterized by loss of cell-cell adhesion of the keratinocytes and, by consequence, skin and mucosal blistering. This condition is caused

Fig. 1.4: (A) electron microscopy image of a desmosome. (B) Schematic arrangement of the proteins

that form the desmosomes. Desmocollin and desmoglein perform Ca2+-dependent cell-cell adhesion via their ectodomain while the C-terminus is placed, on the other side of the plasma membrane, into the outer dense plaque (ODP). Here, the linker proteins plakoglobin (PG), plakophillin (PKP) and desmoplakin (DP) form a complex that connect cadherins with the intermediate filaments (IF). The measurement reported are the distance from the plasma membrane. From21_.

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by the production of antibodies targeting the ectodomain of desmogleins and preventing their homodimerization, thus impairing cell-cell adhesion mediated by desmosomal cadherins.

Other pathologies involving desmosomes are: the staphylococcal scalded skin syndrome, which is due to a toxin produced by staphylococcus that targets desmoglein 1 (the seriousness of the disease depends on the expression of this protein in the infected tissue), the infection by the protozoa Giardia duodenalis and the Bacillus anthracis, and a range of diseases involving mutations on the genes encoding for demosomal cadherins, both neoplasic and non (e.g. arrhythmogenic right ventricular cardiomyopathy).24

Moreover, since desmosomes connect different cells to each other, they also perform an important role in cell signaling.

1.2.3. Communicating junctions

Communicating junctions, which are also commonly referred to as gap junctions, are the most common type of junctions that are present in cells. Contrary to other types of junctions, they do not contribute to the structural integrity of the tissues by physically binding cells together; rather, they connect them at the chemical and electrical level. Gap junctions appear as very dense patches of arrays of channels located in areas where the plasma membranes of adjacent cells are very close by and they are constituted by a wide and conserved family of transmembrane proteins called connexins. These macromolecules organize themselves in hexameric units called connexones, which are basically pores in the cellular membrane. These structures, also called hemichannels, couple with similar ones belonging to a neighboring cell and form the entire channel. Gap junctions allow the passage of inorganic ions and small water-soluble molecules of maximum weight of about 1000 Da between adjacent cells. In this way they provide a metabolic and electrical coupling of the interconnected cells, the latter being implicated in fast signaling pathways. The channel formed by connexins are not always open, but conformational changes may occur so that one or both ends of the hemichannels are closed.25

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1.3. The cadherin superfamily

As described in the previous sections, cadherins are of particular importance in the cell-cell adhesion process. E-cadherin, also known as uvomorulin, was the first calcium-dependent cell adhesion protein to be identified.3_{All the members of the cadherin family}

are characterized by an ectodomain composed by a variable number of Ig-like domains, also referred to as extracellular cadherin domains (ECs). These domains are made of about 110 residues arranged in 7 β-strands forming a "greek key" motif (Fig 1.5).26

The domains are connected in tandem by linker regions containing conserved calcium-binding amino acids and motifs. Calcium provides protein rigidification and is essential for protein function.27

Integral cadherins include also a transmembrane portion, usually a single pass short hydrophobic sequence, and a cytoplasmic domain.

Over time, a wealth of structural and functional information on these proteins has been made available by extensive research programs, thus allowing for an accurate classification of the different members of the cadherin superfamily. Sequence analysis has revealed that there are about 100 different members of this family, each with a specific expression pattern. Although initially done following different and often inconsistent criteria, the sorting of the members of the superfamily eventually converged to a broadly accepted classification that considers E-cadherin as the archetypical member of the family and is based on the sequence and structure similarity analysis of each family member with E-cadherin.

Proteins with high structure and sequence similarity ( 60% or more) to E-cadherin are grouped into the classical cadherin family.28

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This family is further divided into the type I and type II subfamilies because of a small but significant difference in the binding mode, which will be discussed in detail later in this section.29

Owing to a similarity of about 50% with the ectodomain of E-cadherin and their specific localization, cadherins that are found in the desmosomes can be grouped in two different subfamilies, the desmocollins and desmogleins, which differ from each other for the length of their cytoplasmic domain.

The rest of the families, like the so called flamingo, Fat-cadherins and protocadherins families, are composed by proteins with a lower than 50% homology with E-cadherin and, sometimes, have domains that are different from the typical ECs in their ectodomain.30

An analysis of the cadherin superfamily based on sequence similarity of the cytoplasmic domain results in the same classification, once again providing a separation between classical cadherins, desmosomal cadherins and protocadherins. Interestingly, what the cytoplasmic domains of all these different cadherins have in common is a conserved catenin binding region.31

Also in this case, we still have to distinguish between type I by type II classical cadherins, the latter being further divided in subfamilies due to a greater differentiation of the sequence, and as a consequence, the function of the cytoplasmic portion. For desmosomal cadherins, the intracellular domain represents the biggest difference between desmocollins and desmogleins. Finally, in this classification protocadherins are differentiated into clustered, which are further divided into α, β and γ, and non-clustered, based on how their genes are distributed in the genome.32

The cytoplasmic domain of classical cadherins shows two highly conserved regions: a membrane-proximal domain, containing the p120 and δ-catenin binding sites, and a catenin binding domain, with the β-catenin and plakoglobin binding sites.

The classification that is based on the amino acid sequence can be further clarified by genomic analysis.28_{Generally speaking, protein with a small genomic sequence}

difference are close to each other from the evolutionary point of view. Classical and desmosomal cadherins present a very similar organization of the introns, the latter showing great differences in the region encoding for the cytoplasmic domain and precursor peptide and thus accounting for the further classification into desmocollins and desmogleins.

As previously mentioned, protocadherin-encoding genes have their own organization in the genome, and this fact is related both to a different specialization of these proteins and a greater evolutionary distance from the other families.

To evaluate the diversification of these proteins, phylogenetic trees have been calculated, especially on the basis of the sequence encoding for the first extracellular domain, the EC1.28,33,34

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This choice is due to the fact that structural studies, mostly carried out on classical cadherins, clearly shown that the most crucial areas involved in homodimerization, and therefore in adhesion, are located within the EC1.26,27,35–38

High resolution X-ray structure analysis has clearly demonstrated that the mechanism allowing for the formation of cadherin homodimers involves the swap of the first 6 or 7 amino acids of the protein, the A strand of the EC1, and the insertion of the side chain of the Trp 2 residue in a pocket located in the same domain of the partner molecule (strand swap dimer formation). Before crystallography definitively proved this mechanism, mutational studies with chimeric constructs of N- and E-cadherin demonstrated the importance of the EC1 in cadherin homo-dimerization.39

However, to date this binding mode is confirmed only for classical and desmosomal cadherins, while protocadherins are believed to adopt a different mechanism.37,40,41

Owing to the functional importance of the EC1, the amino acid and nucleotide sequence analysis of this domain has been used in order to discriminate and classify different cadherins. One of the largest studies ever done on cadherin sequences was carried out by Hulpiau and coworkers in 2009. In this work both the genes and the amino acid composition of more than 300 members of the superfamily from five different organisms were analyzed.34_{From this phylogenetic study emerged that the superfamily is}

composed by two major branches, the cadherin major branch (CMB) and the cadherin-related major branch (CRMB). The first one is split in two main sub branches, one comprising classical and desmosomal cadherins (collectively referred to as modern cadherins) and the other comprising flamingo, type III and type IV cadherins. The other major branch of the phylogenetic tree is constituted mainly by protocadherins (see fig 1.6).

All the members of the first sub-branch of the CMB have a key Trp in position 2 of the mature protein. This residue plays a crucial role in the dimerization mechanism as it is essential for the swap of the A strand previously mentioned. There are however exceptions: type II cadherins have an additional Trp residue in position 4 which is also involved in the adhesion mechanism, while CDH-13 (T-cadherin), CDH-16 and CDH-17 (7 domains cadherins, 7D) do not have any Trp in their A strand of the EC1 and therefore adopt a different dimerization mechanism.35,42

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Fig. 1.6: phylogenetic tree of the cadherin superfamily. The cadherin major branch is depicted at the top and

the cadherin-related major branch at the bottom. Some subfamilies are grouped in neither of the two major branches. while some cadherin-like proteins have not been completely classified yet. Figure adapted from34_.

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T-cadherin in particular, which has been studied by both NMR and X-ray crystallography, adopts a conformation usually referred to as the x-dimer, which is an intermediate also formed by classical cadherins, where the two monomers adhere at the level of the calcium binding domain between their EC1 and EC2 (fig. 1.7, B).43

Only very limited information is available for the 7D cadherins family. However, it has been demonstrated that their EC3-7 portion is highly homologous to the EC1-5 of classical cadherins, including a Trp in "position 2" of the A strand of their EC3, suggesting, by analogy, that such domain is critically involved in the adhesion function. In fact, experiments performed with LI-cadherin (CDH-17) and E-cadherin (CDH-1) showed trans interaction between the two proteins, likely involving an interaction between the EC3 of one protein and the EC1 of the other.44,45

Proteins of the second sub-branch of CMB have no conserved Trp residues in their EC1. Here we find type III cadherins, which are the non-chordate version of classical cadherins. Members of this family are composed by 13 EC repeats and share a conserved Tyr residue in position 5 of the EC1.

Interestingly, the crystal structure of the EC1 to EC4 fragment shows the protein as a V shaped monomer, a conformation that is also corroborated by analytical ultracentrifugation (AUC) analysis. This is due to the substitution of the calcium binding region between the EC2 and the EC3 with a much more flexible Gly-Gly linker. However, bigger fragments of the ectodomain, such as EC1 to EC9, show clear dimerization, suggesting that these proteins perform their adhesive function by forming relatively big globular structures involving several cadherin repeats.46

Fig. 1.7: schematic representation of the three main conformations that may adopt cadherins. (A)

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In this branch there are also the so-called flamingo cadherins, which are characterized by a seven-pass transmembrane domain and nine EC repeats. Moreover, between these and the plasma membrane there may be globular domains similar to that of laminin A or cystein-rich repeats similar to epithelial growth factor (EGF), a feature that is also shared by some type III cadherins.47

The cadherin-related major branch is composed by four main groups: the largest is composed by the protocadherins, while in the others we find the Fat cadherins, the CDHR-23 (the Cr-2 block in fig. 1.6) family and the Fat-like cadherins.

As previously mentioned, protocadherins are split into the clustered and non-clustered families, which are then further divided into sub-families. Albeit involved in signaling and cell adhesion, these protein do not perform this last function via a strand swapping mechanism, as classical cadherins do, since they lack both a hydrophobic pocket and suitable conserved residues at their N-terminus.

Proteins from the Cr-2 and Cr-3, in particular Cdhr-23 and Pcdh-15, have been studied for their critical mechanotransduction role in hair cells and in those composing the inner ear.

Structural and mutational studies suggest that these two proteins form hetero-tetramers that bind together using specific calcium binding sites located at their N-terminal end, with cis homodimers between proteins protruding from the same cell. This latter interaction mode involves the whole of the large ectodomain of the proteins, 27 ECs for Cdhr-23 and 11 ECs for Pcdh-15.48,49

In conclusion, to completely sort out the members of the promiscuous cadherin superfamily is challenging. However, a thorough phylogenetic analysis accompanied by the analysis the genomic composition and the ectodomain structure, especially of the EC1, allowed for a rather clear definition of the different subfamilies.

Cadherins perform an adhesive function by interacting in a rather specific way, thus proving the extreme degree of specialization they have achieved through evolution. Studies are still ongoing to clarify the mechanisms used by cadherins to bind or discriminate potential partner molecules.

1.3.1. Molecular details of the classical type I cadherin adhesion mechanism

For the purpose of this work, it is particularly interesting to examine the structural data that is available on classical type I cadherins, with a particular focus on the homophilic dimerization mechanism, the topic of the present section.

Over the last three decades, our understanding of the mechanisms involved in cadherin-mediated cell adhesion improved greatly thanks to the continuous increase in the number and the quality of the high resolution crystal structures that have been made available.

A structural approach towards cell adhesion modulation and protein engineering

A STRUCTURAL APPROACH

TOWARDS CELL ADHESION

MODULATION AND PROTEIN

ENGINEERING

Matricola 814045

Coordinatore: prof. Alessio Frassoldati

Tutore: prof. Roberto Piazza

I

Table of contents:

II

III

Index of figures and tables:

His(H)79-IV

V

VI

VII

VIII

IX

X

XII

Abstract

XIII

1

2

1.1.

Cell-cell adhesion

3

4

5

6

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8

1.2. Cell junctions

9

10

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13

1.3.

The cadherin superfamily

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