Alla Gustchina

Structural and biochemical studies of retroviral proteases

Retroviral proteases form a unique subclass of the family of aspartic proteases. These homodimeric enzymes from a number of viral sources have by now been extensively characterized, both structurally and biochemically. The importance of such knowledge to the development of new drugs against AIDS has been, to a large extent, the driving force behind this progress. High-resolution structures are now available for enzymes from human immunodeficiency virus types 1 and 2, simian immunodeficiency virus, feline immunodeficiency virus, Rous sarcoma virus, and equine infectious anemia virus. In this review, structural and biochemical data for retroviral proteases are compared in order to analyze the similarities and differences between the enzymes from different sources and to enhance our understanding of their properties.

Crystal structure of human recombinant interleukin-4 at 2.25 Å resolution

The crystal structure of human recombinant interleukin‐4 (IL‐4) has been solved by multiple isomorphous replacement, and refined to an R factor of 0.218 at 2.25 Å resolution. The molecule is a left‐handed four‐helix bundle with a short stretch of β sheet. The structure bears close resemblance to other cytokines such as granulocyte‐macrophage colony stimulating factor (GM‐CSF). Although no sequence similarity of IL‐4 to GM‐CSF and other related cytokines has been previously postulated, structure‐based alignment of IL‐4 and GM‐CSF revealed that the core of the molecules, including large parts of all four helices and extending over half of the molecule, has 30% sequence identity. This may have identified regions which are not only important to maintain structure, but could also play a role in receptor binding.

The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix.

Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae . Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 and 1.8 Å, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2–34, reveal an unprecedented mode of inhibitor–enzyme interactions. Neither form of the free inhibitor has detectable intrinsic secondary structure in solution. However, upon contact with the enzyme, residues 2–32 become ordered and adopt a near-perfect α-helical conformation. Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity.

Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes.

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0–1.4 Å resolution. The structure of PSCP comprises a single compact domain with a diameter of ∼55 Å, consisting of a seven-stranded parallel β-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab′ fragment of a monoclonal antibody 7C11 was solved at 2.8-Å resolution. Bla g 2 binds to the antibody through four loops that include residues 60-70, 83-86, 98-100, and 129-132. Cation-π interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab′, Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an α-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less β-hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy.

[14]Subsite preferences of retroviral proteinases

Publisher Summary This chapter describes the methodology that has been deployed in the quest for information to facilitate an understanding of the considerable specificity that is exhibited by proteases from different retroviruses. This is followed by an analysis of data from a number of seminal studies. The examples presented in the chapter illustrates several points in which substitution of amino acid side chains leads to the alteration of efficiency in peptide bond cleavage by retroviral proteases. It also emphasizes on the significant role of hydrogen bonding of backbone atoms of the bound substrate to the correct positioning within the active site. The chapter concludes with a discussion on all substrate and inhibitor molecules that form the maximal number of hydrogen bonds, making compromises with the positions of the side chains of the ligand to achieve this.

Slicing a protease : Structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains

ATP‐dependent Lon proteases are multi‐domain enzymes found in all living organisms. All Lon proteases contain an ATPase domain belonging to the AAA+ superfamily of molecular machines and a proteolytic domain with a serine‐lysine catalytic dyad. Lon proteases can be divided into two subfamilies, LonA and LonB, exemplified by the Escherichia coli and Archaeoglobus fulgidus paralogs, respectively. The LonA subfamily is defined by the presence of a large N‐terminal domain, whereas the LonB subfamily has no such domain, but has a membrane‐spanning domain that anchors the protein to the cytoplasmic side of the membrane. The two subfamilies also differ in their consensus sequences. Recent crystal structures for several individual domains and sub‐fragments of Lon proteases have begun to illuminate similarities and differences in structure–function relationships between the two subfamilies. Differences in orientation of the active site residues in several isolated Lon protease domains point to possible roles for the AAA+ domains and/or substrates in positioning the catalytic residues within the active site. Structures of the proteolytic domains have also indicated a possible hexameric arrangement of subunits in the native state of bacterial Lon proteases. The structure of a large segment of the N‐terminal domain has revealed a folding motif present in other protein families of unknown function and should lead to new insights regarding ways in which Lon interacts with substrates or other cellular factors. These first glimpses of the structure of Lon are heralding an exciting new era of research on this ancient family of proteases.

Studies on the role of the S4 substrate binding site of HIV proteinases

Kinetic analysis of the hydrolysis of the peptide H‐Vat‐Ser‐Gin‐Asn‐Tyr*Pro‐He‐Val‐Gin‐NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV‐1 and HIV‐2 proteinases. Deletion of the terminal Val and Gin had only moderate effect on the substrate hydrolysis, while the deletion of the P4, Ser as well as P3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having a high frequency of occurrence in β turns resulted in good substrates, while large amino acids were unfavourable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV‐2 substrate binding pocket.

Comparison of inhibitor binding in HIV-1 protease and in non-viral aspartic proteases: the role of the flap

The crystal structure of HIV‐1 protease with an inhibitor has been compared with the structures of non‐viral aspartic proteases complexed with inhibitors. In the dimeric HIV‐1 protease, two 4‐stranded β‐sheets are formed by half of the inhibitor, residues 27–29, and the flap from each monomer. In the monomeric non‐viral enzyme the single flap does not form a β‐sheet with an inhibitor. The HIV‐1 protease shows more interactions with a longer peptide inhibitor than are observed in non‐viral aspartic protease‐inhibitor complexes. This, and the large movement of the flaps, restricts the conformation of the protease cleavage sites in the retroviral polyprotein precursor.

Crystal structure of the N-terminal domain of E. coli Lon protease.

We report here the first crystal structure of the N‐terminal domain of an A‐type Lon protease. Lon proteases are ubiquitous, multidomain, ATP‐dependent enzymes with both highly specific and non‐specific protein binding, unfolding, and degrading activities. We expressed and purified a stable, monomeric 119‐amino acid N‐terminal subdomain of the Escherichia coli A‐type Lon protease and determined its crystal structure at 2.03 Å (Protein Data Bank [PDB] code 2ANE). The structure was solved in two crystal forms, yielding 14 independent views. The domain exhibits a unique fold consisting primarily of three twisted β‐sheets and a single long α‐helix. Analysis of recent PDB depositions identified a similar fold in BPP1347 (PDB code 1ZBO), a 203‐amino acid protein of unknown function from Bordetella parapertussis, crystallized as part of a structural genomics effort. BPP1347 shares sequence homology with Lon N‐domains and with a family of other independently expressed proteins of unknown functions. We postulate that, as is the case in Lon proteases, this structural domain represents a general protein and polypeptide interaction domain.