A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency.

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 Å resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general.

Generation of human TRIM5alpha mutants with high HIV-1 restriction activity.

Rhesus macaque tripartite motif (TRIM)5alpha potently inhibits early stages of human immunodeficiency virus (HIV)-1 replication, while the human orthologue has little effect on this virus. We used PCR-based random mutagenesis to construct a large library of human TRIM5alpha variants containing mutations in the PRYSPRY domain. We then applied a functional screen to isolate human cells made resistant to HIV-1 infection by the expression of a mutated TRIM5alpha. This protocol led to the characterization of a human TRIM5alpha variant containing a mutation at arginine 335 as conferring resistance to HIV-1 infection. The level of protection stemming from expression of this mutant was comparable to that of previously described mutations at position 332. R332/R335 double mutants decreased permissiveness to HIV-1 and to other lentiviruses by 20- to 50-fold in TE671 fibroblasts and in the T-cell line SUP-T1, and prevented HIV-1 spreading infection as efficiently as the rhesus macaque TRIM5alpha orthologue did. The finding that only two substitutions in human TRIM5alpha can confer resistance to HIV-1 at levels as high as one of the most potent natural orthologues of TRIM5alpha removes a roadblock toward the use of this restriction factor in human gene therapy applications.

Studies of the molecular mechanism of caspase-8 activation by solution NMR.

Caspases are the key players of apoptosis and inflammation. They are present in the cells as latent precursors, procaspases, and are activated upon an apoptotic or inflammatory stimulus. The activation mechanism of caspases has been studied extensively by biochemical and biophysical methods. Additional structural information on active caspases with a variety of different inhibitors bound at the active site is available. In this study, we investigated the cleavage mechanism of caspase-8 from its zymogen to active caspase-8 by solution NMR and by biochemical methods. The intermolecular cleavage reaction using the catalytically inactive C285A procaspase-8 mutant is triggered by adding caspase-8 and followed by (15)N,(1)H-NMR spectroscopy. The spectrum that initially resembles the one of procaspase-8 gradually over time changes to that of caspase-8, and disappearing peaks display exponential decaying intensities. Removal of either one of the cleavage recognition motifs in the linker, or phosphorylation at Tyr380, is shown to reduce the rate of the cleavage reaction. The data suggest that dimerization repositions the linker to become suitable for intermolecular processing by the associated protomer. Furthermore, analysis of inhibitor binding to the active caspase-8 reveals an induced-fit mechanism for substrate binding.

nDsbD: a redox interaction hub in the Escherichia coli periplasm.

DsbD is a redox-active protein of the inner Escherichia coli membrane possessing an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain. nDsbD interacts with four different redox proteins involved in the periplasmic disulfide isomerization and in the cytochrome c maturation systems. We review here the studies that led to the structural characterization of all soluble DsbD domains involved and, most importantly, of trapped disulfide intermediate complexes of nDsbD with three of its four redox partners. These results revealed the structural features enabling nDsbD, a 'redox hub' with an immunoglobulin-like fold, to interact efficiently with its different thioredoxin-like partners.

Zooming in on the hydrophobic ridge of H-2D(b): implications for the conformational variability of bound peptides.

Class I major histocompatibility complex (MHC) molecules, which display intracellularly processed peptides on the cell surface for scanning by T-cell receptors (TCRs), are extraordinarily polymorphic. MHC polymorphism is believed to result from natural selection, since individuals heterozygous at the corresponding loci can cope with a larger number of pathogens. Here, we present the crystal structures of the murine MHC molecule H-2D(b) in complex with the peptides gp276 and np396 from the lymphocytic choriomeningitis virus (LCMV), solved at 2.18 A and 2.20 A resolution, respectively. The most prominent feature of H-2D(b) is a hydrophobic ridge that cuts across its antigen-binding site, which is conserved in the L(d)-like family of class I MHC molecules. The comparison with previously solved crystal structures of peptide/H-2D(b) complexes shows that the hydrophobic ridge focuses the conformational variability of the bound peptides in a "hot-spot", which could allow optimal TCR interaction and discrimination. This finding suggests a functional reason for the conservation of this structural element.

Structure of the soluble domain of a membrane-anchored thioredoxin-like protein from Bradyrhizobium japonicum reveals unusual properties.

TlpA is an unusual thioredoxin-like protein present in the nitrogen-fixing soil bacterium Bradyrhizobium japonicum. A hydrophobic N-terminal transmembrane domain anchors it to the cytoplasmic membrane, whereby the hydrophilic thioredoxin domain becomes exposed to the periplasmic space. There, TlpA catalyses an essential reaction, probably a reduction, in the biogenesis of cytochrome aa(3). The soluble thioredoxin domain (TlpA(sol)), devoid of the membrane anchor, was purified and crystallized. Oxidized TlpA(sol) crystallized as a non-covalent dimer in the space group P2(1)2(1)2(1). The X-ray structure analysis was carried out by isomorphous replacement using a xenon derivative. This resulted in a high-resolution (1.6 A) three-dimensional structure that displayed all of the features of a classical thioredoxin fold. A number of peculiar structural details were uncovered: (i) Only one of the two active-site-cysteine sulphurs (Cys72, the one closer to the N terminus) is exposed on the surface, making it the likely nucleophile for the reduction of target proteins. (ii) TlpA(sol) possesses a unique structural disulphide bond, formed between Cys10 and Cys155, which connects an unprecedented N-terminal alpha helix with a beta sheet near the C terminus. (iii) An insertion of about 25 amino acid residues, not found in the thioredoxin prototype of Escherichia coli, contributes only marginally to the thioredoxin fold, but forms an extra, surface-exposed alpha helix. This region plus another surface-exposed stretch (-Ile-Gly-Arg-Ala-), which is absent even in the closest TlpA relatives, might be considered as specificity determinants for the recognition of target proteins in the periplasm. The TlpA(sol) structure paves the way towards unraveling important structure-function relationships by rational mutagenesis.

Crystal structure of neuroserpin: a neuronal serpin involved in a conformational disease.

The protease inhibitor neuroserpin regulates the development of the nervous system and its plasticity in the adult. Neuroserpins carrying the Ser53Pro or Ser56Arg mutation form polymers in neuronal cells. We describe here the structure of wild-type neuroserpin in a cleaved form. The structure provides a basis to understand the role of the mutations in the polymerization process. We propose that these mutations could delay the insertion of the reactive center loop into the central beta-sheet A, an essential step in the inhibition and possibly in the polymerization of neuroserpin.

Structural genomics: opportunities and challenges.

Following the complete genome sequencing of an increasing number of organisms, structural biology is engaging in a systematic approach of high-throughput structure determination called structural genomics to create a complete inventory of protein folds/structures that will help predict functions for all proteins. First results show that structural genomics will be highly effective in finding functional annotations for proteins of unknown function.

Caspases: key players in programmed cell death.

Research in apoptosis has established a central role for caspases. The recent determination of structures of caspase-1, caspase-3 and caspase-8, together with biochemical studies, has greatly enhanced our understanding of the structure, function and specificity of these enzymes. This provides a basis for the further elucidation of the biological role of caspases and a guide to the design of selective inhibitors to treat caspase-mediated diseases.

Viral escape at the molecular level explained by quantitative T-cell receptor/peptide/MHC interactions and the crystal structure of a peptide/MHC complex.

Viral escape, first characterized for the lymphocytic choriomeningitis virus (LCMV) in a mouse transgenic for the P14 T cell-receptor (TCR), can be due to mutations in T-cell epitopes. We have measured the affinity between the H-2D(b) containing the wild-type and two of its "viral escape" epitopes, as well as other altered peptide ligands (APL), by using BIACORE analysis, and solved the crystal structure of H-2D(b) in complex with the wild-type peptide at 2.75 A resolution. We show that viral escape is due to a 50 to 100-fold reduction in the level of affinity between the P14 TCR and the binary complexes of the MHC molecule with the different peptides. Structurally, one of the mutations alters a TCR contact residue, while the effect of the other on the binding of the TCR must be indirect through structural rearrangements. The former is a null ligand, while the latter still leads to some central tolerance. This work defines the structural and energetic threshold for viral escape.

Caspase-8 specificity probed at subsite S(4): crystal structure of the caspase-8-Z-DEVD-cho complex.

Caspase-8 is an initiator enzyme in the Fas-mediated pathway of which the downstream executioner caspase-3 is a physiological target. Caspases are cysteine proteases that are specific for substrates with an aspartic acid residue at the P(1) position and have an optimal recognition motif that incorporates four amino acid residues N-terminal to the cleavage site. Caspase-8 has been classified as a group III caspase member because it shows a preference for a small hydrophobic residue at the P(4) substrate position. We report the X-ray crystallographic structure of caspase-8 in complex with benzyloxycarbonyl-Asp-Glu-Val-Asp-aldehyde (Z-DEVD), a specific group II caspase inhibitor. The structure shows that the inhibitor interacts favourably with the enzyme in subsite S(4). Kinetic data reveal that Z-DEVD (K(i) 2 nM) is an almost equally potent inhibitor of caspase-8 as the specific group III inhibitor Boc-IETD-aldehyde (K(i) 1 nM). In view of this finding, the original classification of caspases into three specificity groups needs to be modified, at least for caspase-8, which tolerates small hydrophobic residues as well as the acidic residue Asp in subsite S(4). We propose that the subsite S(3) must be considered as an important specificity-determining factor.

Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin.

BACKGROUND: The aspartic proteinase renin plays an important physiological role in the regulation of blood pressure. It catalyses the first step in the conversion of angiotensinogen to the hormone angiotensin II. In the past, potent peptide inhibitors of renin have been developed, but none of these compounds has made it to the end of clinical trials. Our primary aim was to develop novel nonpeptide inhibitors. Based on the available structural information concerning renin-substrate interactions, we synthesized inhibitors in which the peptide portion was replaced by lipophilic moieties that interact with the large hydrophobic S1/S3-binding pocket in renin. RESULTS: Crystal structure analysis of renin-inhibitor complexes combined with computational methods were employed in the medicinal-chemistry optimisation process. Structure analysis revealed that the newly designed inhibitors bind as predicted to the S1/S3 pocket. In addition, however, these compounds interact with a hitherto unrecognised large, distinct, sub-pocket of the enzyme that extends from the S3-binding site towards the hydrophobic core of the enzyme. Binding to this S3(sp) sub-pocket was essential for high binding affinity. This unprecedented binding mode guided the drug-design process in which the mostly hydrophobic interactions within subsite S3(sp) were optimised. CONCLUSIONS: Our design approach led to compounds with high in vitro affinity and specificity for renin, favourable bioavailability and excellent oral efficacy in lowering blood pressure in primates. These renin inhibitors are therefore potential therapeutic agents for the treatment of hypertension and related cardiovascular diseases.

The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase.

Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned. Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a stringent pattern of cysteine residues. The H. pylori cysteine-rich protein A (HcpA), which is a member of this family, was expressed and refolded from inclusion bodies. Six pairs of cysteine residues, which are separated by exactly seven residues, form disulfide bridges. HcpA is a beta-lactamase. It slowly hydrolyzes 6-aminopenicillinic acid and 7-aminocephalosporanic acid (ACA) derivatives. The turnover for 6-aminopenicillinic acid derivatives is 2-3 times greater than for ACA derivatives. The enzyme is efficiently inhibited by cloxacillin and oxacillin but not by ACA derivatives or metal chelators. We suggest that all family 12 members possess similar activities and might be involved in the synthesis of the cell wall peptidoglycan. They might also be responsible for amoxicillin resistance of certain H. pylori strains.

The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure.

The question of whether a protein whose natural sequence is inverted adopts a stable fold is still under debate. We have determined the 2. 1-A crystal structure of the retro-GCN4 leucine zipper. In contrast to the two-stranded helical coiled-coil GCN4 leucine zipper, the retro-leucine zipper formed a very stable, parallel four-helix bundle, which now lends itself to further structural and functional studies.

The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis.

BACKGROUND: In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to the cell receptor as a zymogen (procaspase-8) and is incorporated into the death-signalling complex. Procaspase-8 is subsequently activated leading to a cascade of proteolytic events, one of them being the activation of caspase-3, and ultimately resulting in cell destruction. Variations in the substrate specificity of different caspases have been reported. RESULTS: We report here the crystal structure of a complex of the activated human caspase-8 (proteolytic domain) with the irreversible peptidic inhibitor Z-Glu-Val-Asp-dichloromethylketone at 2.8 A resolution. This is the first structure of a representative of the long prodomain initiator caspases and of the group III substrate specificity class. The overall protein architecture resembles the caspase-1 and caspase-3 folds, but shows distinct structural differences in regions forming the active site. In particular, differences observed in subsites S(3), S(4) and the loops involved in inhibitor interactions explain the preference of caspase-8 for substrates with the sequence (Leu/Val)-Glu-X-Asp. CONCLUSIONS: The structural differences could be correlated with the observed substrate specificities of caspase-1, caspase-3 and caspase-8, as determined from kinetic experiments. This information will help us to understand the role of the various caspases in the propagation of the apoptotic signal. The information gained from this investigation should be useful for the design of specific inhibitors.

The 1.2 A crystal structure of hirustasin reveals the intrinsic flexibility of a family of highly disulphide-bridged inhibitors.

BACKGROUND: Leech-derived inhibitors have a prominent role in the development of new antithrombotic drugs, because some of them are able to block the blood coagulation cascade. Hirustasin, a serine protease inhibitor from the leech Hirudo medicinalis, binds specifically to tissue kallikrein and possesses structural similarity with antistasin, a potent factor Xa inhibitor from Haementeria officinalis. Although the 2.4 A structure of the hirustasin-kallikrein complex is known, classical methods such as molecular replacement were not successful in solving the structure of free hirustasin. RESULTS: Ab initio real/reciprocal space iteration has been used to solve the structure of free hirustasin using either 1.4 A room temperature data or 1.2 A low temperature diffraction data. The structure was also solved independently from a single pseudo-symmetric gold derivative using maximum likelihood methods. A comparison of the free and complexed structures reveals that binding to kallikrein causes a hinge-bending motion between the two hirustasin subdomains. This movement is accompanied by the isomerisation of a cis proline to the trans conformation and a movement of the P3, P4 and P5 residues so that they can interact with the cognate protease. CONCLUSIONS: The inhibitors from this protein family are fairly flexible despite being highly cross-linked by disulphide bridges. This intrinsic flexibility is necessary to adopt a conformation that is recognised by the protease and to achieve an optimal fit, such observations illustrate the pitfalls of designing inhibitors based on static lock-and-key models. This work illustrates the potential of new methods of structure solution that require less or even no prior phase information.

Crystallization and structure solution of p53 (residues 326-356) by molecular replacement using an NMR model as template.

The molecular replacement method is a powerful technique for crystal structure solution but the use of NMR structures as templates often causes problems. In this work the NMR structure of the p53 tetramerization domain has been used to solve the crystal structure by molecular replacement. Since the rotation- and translation-functions were not sufficiently clear, additional information about the symmetry of the crystal and the protein complex was used to identify correct solutions. The three-dimensional structure of residues 326-356 was subsequently refined to a final R factor of 19.1% at 1.5 A resolution.

Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone.

The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity. The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 A. The asymmetric unit contains a (p17/p12)2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography. The overall topology of CPP32 is very similar to that of interleukin-1beta-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32. A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site. In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor. The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE. This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition. This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.

Characterization, crystallization and preliminary crystallographic analysis of human recombinant cyclooxygenase-2.

Purified recombinant membrane apoprotein cyclooxygenase-2 (COX-2) has been reconstituted with heme and characterized. The holoprotein has been crystallized in complex with the selective inhibitor CGP 28238 [6-(2,4-difluorophenoxy)-5- methylsulfonylamino-l-indanone] by the sitting-drop method of vapor diffusion using polyethylene glycol 2000 monomethyl ether as precipitant, in the presence of the nonionic detergent beta-octylglucoside. The crystals are orthorhombic, belonging to the space group P2(1)2(1)2 with cell dimensions a = 209.56, b = 71.28 and c = 93.82 A, and diffract to 2.5 A resolution. The asymmetric unit contains two COX-2 monomers, as confirmed by the molecular replacement solution and in agreement with the dimeric structure of the detergent-solubilized protein found with dynamic light scattering and size-exclusion chromatography. Structural work is in progress.