Structural & Supramolecular Chemistry Research Group – Research – Structure analysis of biological macromolecules

C. Structure analysis of biological macromolecules

C1. Introduction: X-ray crystallography of biological macromolecules

X-ray crystallography allows a “look” at the detailed structure of molecules in 3 dimensions. This is the reason why it had an immense impact in chemistry, material science and biology. It has generated a new field in the latter, i.e. “Structural Biology”. Knowledge of the structure of biological macromolecules can explain their function (or malfunction), reveal mechanisms and pathways in the cells and eventually allow us to understand life and use this knowledge to our benefit, e.g. to improve the design of drugs.

Past projects include:C1-C5 Glucogen Phosphorylase b with γ-CD,C1 several 2[4Fe4S] ferredoxins from pathogenic bacteria,C2-3 certain domains of the muscle proteins titinC4 and myomesin.C5

Figure C1. (a) γ-Cyclodextrin (in red) binds to the storage site of the Glucose Phosphorylase b dimer; (b) Ribbon representation of the Pheudomonas aeruginosa ferredoxin; (c) Ribbon representation of the two antiparallel titin domains Z1 (blue) and Z2 (cyan including the Z1-Z2 linker) cross-linked by telethonin protein (red).

C2. Endoplasmic Reticulum Aminopeptidases

Endoplasmic Reticulum (ER) aminopeptidases ERAP1, ERAP2 and IRAP cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding onto Major Histocompatibility Complex class I (MHCI) molecules and help regulate the adaptive immune response. In collaboration with the group of E. Stratikos at our institution, we have determined for the first time the structure of ERAP2 to 3.08 Å by X-ray crystallography.C6 The ERAP2 structure provides a structural explanation for the different peptide N-terminus specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property to their biological role.

Figure C2. Ribbon representation of ERAP2

Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. The common ERAP2 SNP rs2549782, codes for amino acid variation N392K that leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared to the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. X-ray crystallographic analysis of the 392K allele of ERAP2 suggests that the polymorphism interferes with the stabilization of the N-terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis.C7 The study provides mechanistic insight to the association of this ERAP2 polymorphism with disease and supports the idea that polymorphic variation in antigen processing enzymes constitutes a component of immune response variability in humans.

The structure of antigen-processing aminopeptidase ERAP2 in complex with phosphinic pseudopeptide inhibitors is also being studied (Figure C3). Thus the structure of an ERAP2/pseudotripeptide complex has been determinedC8, as well as that of an ERAP2/pseudodecapeptide complex.C9

Figure C3. (a) A phosphinic pseudotripeptide inhibitor bound in the ERAP2 catalytic site; (b) ERAP2 (colored by domain) complexed with pseudodecapeptide substrate analogue (in red); (c) ERAP2 internal cavity (in surface representation) with complexed pseudodecapeptide.

Figure C4. Structure of the IRAP/pseudodecapeptide complex. Structural domains are color coded and named. The pseudodecapeptide ligand is shown as yellow spheres, and the active site Zn(II) atom as a red sphere.

The structure of another aminopeptidase, Insulin-Regulated Aminopeptidase (IRAP), an enzyme involved in antigen preparation for presentation to the immune system has also been solved both free of substrate and bound with the previously mentioned pseudodecapeptide (Figure C4).C10

The project has now entered a new phase, i.e. rationally designed inhibitors (drug candidates). By co-crystallizing the ERAP1, ERAP2 and IRAP proteins with various inhibitors and solving their crystal structures, the binding behavior of the inhibitors to the protein, as determined by biophysical and biochemical methods, can be explained by the structural characteristics of the protein-inhibitor complex (Figure C5). Thus insight is gained on the structural requirements that an inhibitor should have in order to have a better (or the best) binding affinity to the protein, which can provide the lead drug candidate.C11-12

 
 Figure C5. ERAP2 internal cavity (in surface representation) occupied by three inhibitors: 3,4-diaminobenzoic acid (green) hydroxamic acid (blue) and pseudotripeptide (red). Comparison of the crystal structures of the first two inhibitors with the potent phosphinic pseudotripeptide inhibitor of ERAP2 in complex, suggests that engaging the substrate N-terminus recognition properties of the active site is crucial for inhibitor binding even in the absence of a potent zinc-binding group.

C3. DsbD: a bacterial thiol-disulfide oxidoreductase 

DsbD, one of the five proteins of the bacterial Disulfide bond (Dsb) formation system, is an accessory protein to the bacterial Cytochrome c maturation (Ccm) system. Three structures of the soluble domains of the transmembrane protein DsbD have been solved, i.e. the N-terminal domain in the reduced formC13 and the C-terminal domains of two point-mutant variants.C14 DsbD is located in the inner membrane of Gram-negative bacteria and it is responsible for transporting reducing power from the cytoplasm to the oxidising periplasm of the cell. It comprises three domains: the central transmembrane domain of unknown structure (tmDsbD) is flanked by two globular periplasmic domains, the N-terminal domain (nDsbD) with an immunoglobulin-like fold and the C-terminal domain (cDsbD) with a classical thioredoxin (Trx) fold. DsbD plays an important role in oxidative protein folding because it allows for the correct formation of disulfide bonds in proteins functioning in harsh extracytoplasmic environments. It is also involved in bacterial pathogenesis as the expression and stability of most virulent factors (secreted molecules, secretion apparati, adhesion systems etc.) are dependent on the presence of DsbD. The studies are in collaboration with the Department of Biochemistry, University of Oxford (Prof. Stuart J. Ferguson, Prof. Christina Redfield and Dr. Despoina A. I. Mavridou). This work contributed significantly in the elucidation of the interactions of the soluble domains of this unique oxidoreductase, but also in the general understanding of the factors controlling the reactivity of the ubiquitous thioredoxin fold.

  1. The binding of beta and gamma-cyclodextrins to glycogen phosphorylase b: kinetic and crystallographic studies. Pinotsis, D. D. Leonidas, E. D. Chrysina, N. G. Oikonomakos, I. M. Mavridis Protein Sci. 2003, 12, 1914-1924.
  2. The structure of the 2[4Fe-4S] ferredoxin from Pseudomonas aeruginosa at 1.32 Å resolution. Comparison with other high resolution structures of ferredoxins and contributing structural features to reduction potential values. Giastas, N. Pinotsis, G. Efthymiou, M. Wilmanns, P. Kyritsis, J-M. Moulis, I. M. Mavridis J. Biolog. Inorg. Chem. 2006, 11, 445-458.
  3. Insight into the protein and solvent contributions to the reduction potentials of [4Fe-4S]2+/+ clusters: Crystal structures of the Allochromatium vinosum ferredoxin variants C57A and V13G and the homologous Escherichia coli ferredoxin. Saridakis, P. Giastas, G. Efthymiou, V. Thoma, J-M. Moulis, P. Kyritsis, I. M. Mavridis J. Biolog. Inorg. Chem. 2009, 14, 783-799.
  4. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. P. Zou, N. Pinotsis, S. Lange, Y-H. Song, A. Popov, Mavridis, O. M. Mayans, M. Gautel, M. Wilmanns Nature 2006, 439, 225-228.
  5. Superhelical architecture of the myosin filament-linking protein myomesin with unusual elastic properties. Pinotsis, S. D. Chatziefthimiou, F. Berkemeier, F. Beuron, I. M. Mavridis, P. V. Konarev, D. I. Svergun, E. Morris, M. Rief, M. Wilmanns, PLoS Biol. 2012, Feb;10(2):e1001261.
  6. Crystal Structure of Human ER Aminopeptidase 2 Reveals Atomic Basis for Distinct Roles in Antigen Processing. R. Birtley, E. Saridakis, E. Stratikos, M. Mavridis Biochemistry, 2012, 51, 286-295.
  7. A Common Single Nucleotide Polymorphism in Endoplasmic Reticulum Aminopeptidase 2 Induces a Specificity Switch that Leads to Altered Antigen Processing. D. Evnouchidou, J. Birtley, S. Seregin, A. Papakyriakou, E. Zervoudi, M. Samiotaki, G. Panayotou, P. Giastas, O. Petrakis, D. Georgiadis, A. Amalfitano, Saridakis, I. M. Mavridis, E. Stratikos, J. Immunol. 2012, 189, 2383-2392.
  8. A rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses. E. Zervoudi, E. Saridakis, J.R. Birtley, S. Seregin , E. Reeves, P. Kokkala, Y.A. Aldhamen, A. Amalfitano, I.M. Mavridis, E. James, D. Georgiadis and E. Stratikos, Natl. Acad. Sci. USA 2013, 110, 19890-19895.
  9. Structural basis for antigenic peptide recognition and processing by ER aminopeptidase 2. A. Mpakali, P. Giastas, N. Mathioudakis, I.M. Mavridis, E. Saridakis, E. Stratikos J. Biol. Chem. 2015, 290, 26021-26032.
  10. Crystal structure of Insulin-Regulated Aminopeptidase with bound substrate analogue provides insight on antigenic epitope precursor recognition and processing. Mpakali, E. Saridakis, K. Harlos, Y. Zhao, A. Papakyriakou, P. Kokkala, D. Georgiadis, E. Stratikos, J. Immunol. 2015, 195, 2842-2851.
  11. “Crystal Structures of ERAP2 Complexed with Inhibitors Reveal Pharmacophore Requirements for Optimizing Inhibitor Potency” A. Mpakali, P. Giastas, R. Deprez-Poulain, A. Papakyriakou, D. Koumantou, R. Gealageas, S. Tsoukalidou, D. Vourloumis, I. M Mavridis, E. Stratikos, E. Saridakis, ACS Medicinal Chemistry Letters 2017, 8 (3), 333-337.
  12. “Ligand-induced conformational change of Insulin-regulated aminopeptidase: insights on catalytic mechanism and active site plasticity” A. Mpakali, E. Saridakis, K. Harlos, Y. Zhao, P. Kokkala, D. Georgiadis, P. Giastas, A. Papakyriakou, E. Stratikos, Journal of Medicinal Chemistry 2017, 60, 2963-2972.
  13. Oxidation-state-dependent protein-protein interactions in disulfide cascades. D. A. I. Mavridou, E. Saridakis, P. Kritsiligkou, A.D. Goddard, J.M. Stevens, S.J. Ferguson and C. Redfield. Biol. Chem. 2011, 286, 24943-24956.
  14. An extended active-site motif controls the reactivity of the thioredoxin fold. D. A. I. Mavridou, E. Saridakis, P. Kritsiligkou, E.C. Mozley, S.J. Ferguson and C. Redfield, Biol. Chem. 2013, 289, 8681-8696.