Antibody-antigen interactions: new structures and new conformational changes

Yet Another Numbering Scheme for Immunoglobulin Variable Domains: An Automatic Modeling and Analysis Tool

One way to combat infectious diseases is to selectively inhibit foreign proteases within host cells, thus retarding replication rates of infectious organisms and assisting normal immunological defense mechanisms involved in their eradication. This review gives a summary of over 1500 three dimensional crystal (X-ray) and solution (NMR) structures from the pdb of substrates, products and inhibitors bound in the active sites of aspartic, serine, metallo, cysteine, and threonine endopeptidases. These active sites of all five protease classes recognize peptidic and non-peptidic ligands in an extended beta strand conformation, with few exceptions. Comparisons of protease-bound ligand conformations are illustrated by structural superpositions for a subset of structures, including 21 aspartic, 44 serine, 20 metallo, 23 cysteine, and 2 threonine proteases, among the protease-ligand structures analyzed. The extended substrate-binding mode is also illustrated for 3 aspartic proteases, 1 serine protease, 1 cysteine protease and 1 metalloprotease.

Proteases universally recognize beta strands in their active sites.

Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering.

https://www.cell.com/structure/pdf/S0969-2126(02)00720-7.pdf

Substrate shape determines specificity of recognition for HIV-1 protease: analysis of crystal structures of six substrate complexes.

NMR and crystallography have been used to study antigen conformational changes that occur in a trisaccharide-Fab complex in solution and in the solid state. NOE buildup rates from transferred NOE experiments show that the antigenic determinant of a Salmonella lipopolysaccharide, represented by the trisaccharide methyl glycoside alpha-D-Galp(1-->2 [alpha-D-Abep(1-->3)]- alpha-D-Manp1-->OMe (1), undergoes a protein-induced conformational shift about the Gal-->Man glycosidic linkage when it is bound by a monoclonal antibody in aqueous solution. The same trisaccharide was crystallized with Fab, and a solved structure at 2.1-A resolution revealed that the conformation of the trisaccharide ligand was similar to that seen in a dodesaccharide-Fab complex [Cygler et al. (1991) Science 253, 442-445), where the Gal-Man linkage also experienced a similar conformational shift. Distance constraints derived from the TRNOE buildup curves are consistent with two bound trisaccharide conformations, one of which correlates with the ligand conformation of the crystalline Fab-trisaccharide complex. In this bound conformation, short interatomic distances between Abe O-2 and Gal O-2 permit an oligosaccharide intramolecular hydrogen bond. Despite its relatively low energy, a preponderance of this conformer could not be detected in aqueous or DMSO solutions of free trisaccharide by either 1H or 13C NMR experiments. In DMSO, a different intramolecular hydrogen bond between Abe O-2 and Man O-4 was observed due to a solvent-induced shift in the conformational equilibria (relative to aqueous solution). Molecular modeling of the trisaccharide in the binding site and as the free ligand suggested that the protein imposes an induced fit on the antigen, primarily resulting in a shift of the Gal-Man phi torsional angle. This reduces the interproton separation between Abe H-3 and Gal H-1 with a marked increase in the intensity of the previously weak NOEs between the protons of the noncovalently linked galactose and abequose residues. The impact of the conformational shift on gross trisaccharide topology is sufficiently small that binding modes inferred from functional group replacements are not impaired.

Solution structure of a trisaccharide-antibody complex: comparison of NMR measurements with a crystal structure.

Crystal structures of complexes of a D30N mutant of feline immunodeficiency virus protease (FIV PR) complexed with a statine-based inhibitor (LP-149), as well as with a substrate based on a modification of this inhibitor (LP-149S), have been solved and refined at resolutions of 2.0 and 1.85 A, respectively. Both the inhibitor and the substrate are bound in the active site of the mutant protease in a similar mode, which also resembles the mode of binding of LP-149 to the native protease. The carbonyl oxygen of the scissile bond in the substrate is not hydrated and is located within the distance of a hydrogen bond to an amido nitrogen atom from one of the two asparagines in the active site of the enzyme. The nitrogen atom of the scissile bond is 3.25 A from the conserved water molecule (Wat301). A model of a tetrahedral intermediate bound to the active site of the native enzyme was built by considering the interactions observed in all three crystal structures of FIV PR. Molecular dynamics simulations of this model bound to native wild-type FIV PR were carried out, to investigate the final stages of the catalytic mechanism of aspartic proteases.

https://mcl1.ncifcrf.gov/wlo_pubs/162.pdf

Crystal Structures of the Inactive D30N Mutant of Feline Immunodeficiency Virus Protease Complexed with a Substrate and an Inhibitor

N alpha-Acetyl[D-Phe45,Arg47]hirudin45-65 (P53) is a bivalent thrombin inhibitor (Ki = 56 nM) that consists of an active site inhibitor segment, [N alpha-acetyl-(dF)PRP]; a fibrinogen recognition exo site inhibitor segment, hirudin55-65 (DFEEIPEEYLQ-OH); and a linker, hirudin49-54 (QSHNDG), connecting these inhibitor segments (DiMaio et al, 1990) The structure-function relationships of the linker were studied using a combination of various omega-amino acids, which modified the length of the linker as well as the number and the locations of peptide bonds Linkers with 14-18 atoms (counting only the atoms contributing to the length of the linker) showed a competitive inhibition with Ki = 17-34 nM The potency of the inhibitors with 12-13-atom linkers was sensitive to the chemical structure of the linker The high-potency inhibitors showed a competitive inhibition, while the low-potency inhibitors showed a hyperbolic inhibition Among them, an inhibitor with a 13-atom linker showed the highest potency (Ki = 051 nM, an 11-fold improvement from that of P53 above), indicating that this is an optimal linker length Since linkers with 6-10 atoms failed to bridge the active site and exo site inhibitor segments, a minimum of 11 atoms was required to bridge them, even though the potency of the inhibitor with an 11-atom linker was weak (Ki = 26 nM) Molecular dynamics simulation of the inhibitors with 13-atom linkers suggested that some linkers serve as a functional domain with the amide bond of the linker interacting with thrombin through hydrogen bonds(ABSTRACT TRUNCATED AT 250 WORDS)

Alexander Zdanov

Papers

Solution structure of a trisaccharide-antibody complex: comparison of NMR measurements with a crystal structure.

Crystal Structures of the Inactive D30N Mutant of Feline Immunodeficiency Virus Protease Complexed with a Substrate and an Inhibitor

Design of a linker for trivalent thrombin inhibitors: interaction of the main chain of the linker with thrombin.