Structure-based secondary structure-independent approach to design protein ligands: Application to the design of Kv1.2 potassium channel blockers.

We have developed a structure-based approach to the design of protein ligands. This approach is based on the transfer of a functional binding motif of amino acids, often referred as to the "hot spot", on a host protein able to reproduce the functional topology of these residues. The scaffolds were identified by a systematic in silico search in the Protein Data Bank for proteins possessing a group of residues in a topology similar to that adopted by the functional motif in a reference ligand of known 3D structure. In contrast to previously reported studies, this search is independent of the particular secondary structure supporting the functional motif. To take into account the global properties of the host protein, two additional criteria were taken into account in the selection process: (1) Only those scaffolds sterically compatible with the positioning of the functional motif as observed in a reference complex model were retained. (2) Host proteins displaying electrostatic potentials, in the region of the transferred functional motif, similar to that of the reference ligand were selected. This approach was applied to the development of protein ligands of the Kv1.2 channel using BgK, a small protein isolated from the sea anemone Bunodosoma granulifera, as the reference ligand. Four proteins obtained by this approach were produced for experimental evaluation. The X-ray structure of one of these proteins was determined to check for similarity of the transferred functional motif with the structure it adopts in the reference ligand. Three of these protein ligands bind the Kv1.2 channel with inhibition constants of 0.5, 1.5, and 1.6 microM. Several mutants of these designed protein ligands gave binding results consistent with the presumed binding mode. These results show that protein ligands can be designed by transferring a binding motif on a protein host selected to reproduce the functional topology of this motif, irrespective to the secondary structure supporting the functional motif, if the host protein possesses steric and electrostatic properties compatible with the binding to the target. This result opens the way to the design of protein ligands by taking advantage of the considerable structural repertoire of the Protein Data Bank.

Highly specific anti-estradiol antibodies: structural characterisation and binding diversity.

Subtle modulation of antibody-binding properties by protein engineering often lies with an accurate structural and energetic description of how an antigen is recognised. Thus, with the intent to increase the affinity and add a bias in favour of natural estradiol compared with its chemically modified immunogen, we have determined the crystal structure of two anti-estradiol monoclonal antibodies, 10G6D6 and 17E12E5. Although generated against the same estradiol derivative, these antibodies share little sequence identity, which is reflected in dissimilar binding pockets and in different positioning of the steroid. In both antibodies the characteristic 17-hydroxyl group is buried deeply at the bottom of hydrophobic pockets and stabilised by hydrogen bonds. Apart from this similarity, the steroid is oriented differently in the respective binding pockets. The high specificity of both antibodies has been mapped out, and even closely related steroids show low cross-reactivity. The structural studies of the complex formed between 10G6D6 and 6-CMO-estradiol have identified contacts between the 6-CMO coupling linker and an arginine residue from the heavy chain CDR2 segment. This segment is now being targeted by random mutagenesis to select mutants with a preference for natural estradiol compared to the branched hapten.

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.

Design of a Gag pentapeptide analogue that binds human cyclophilin A more efficiently than the entire capsid protein: new insights for the development of novel anti-HIV-1 drugs.

Cyclophilin A (hCyp-18), a ubiquitous cytoplasmic peptidyl-prolyl cis/trans isomerase (PPIase), orchestrates HIV-1 core packaging. hCyp-18, incorporated into the virion, enables core uncoating and RNA release and consequently plays a critical role in the viral replication process. hCyp-18 specifically interacts with a single exposed loop of the Gag polyprotein capsid domain via a network of nine hydrogen bonds which mainly implicates a 7-mer fragment of the loop. As previously reported, the corresponding linear heptapeptide Ac-Val-His-Ala-Gly-Pro-Ile-Ala-NH(2) (2) binds to hCyp-18 with a low affinity (IC(50) = 850 +/- 220 microM) but a potentially useful selectivity for hCyp-18 relative to hFKBP-12, another abundant PPIase. On the basis of X-ray structures of Gag fragments:hCyp-18 complexes, we generated a series of modified peptides in order to probe the determinants of the interaction and hence to select a peptidic ligand displaying a higher affinity than the capsid domain of Gag. We synthesized a series of heptapeptides to test the energetic contribution of amino acids besides the Gly-Pro moiety. In particular the importance of the histidine residue for the interaction was underscored. We also investigated the influence of N- and C-terminal modifications. Hexapeptides containing either deaminovaline (Dav) in place of the N-terminal valine or substitution of the C-terminal alanine amide with a benzylamide group displayed increased affinities. Combination of both modifications gave the most potent competitor Dav-His-Ala-Gly-Pro-Ile-NHBn (28) which has a higher affinity for hCyp-18 (K(d) = 3 +/- 0.5 microM) than the entire capsid protein (K(d) = 16 +/- 4 microM) and a very low affinity for hFKBP-12. Some of our results strongly suggest that the title compound is not a substrate of hCyp-18 and interacts preferentially in the trans conformation.

Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity.

Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

On the role of the cis-proline residue in the active site of DsbA.

In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis-proline in proximity of the active site residues. In the variant DsbA(P151A), this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild-type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbA(P151A) crystallized in a different crystal form from the wild-type protein, in space group P2(1) with six molecules in the asymmetric unit. Its X-ray structure was determined to 2.8 A resolution. The most significant conformational changes occurred at the active site. The loop 149-152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the asymmetric unit a gauche conformation not observed in the wild-type protein. The X-ray structure and folding studies on DsbA(P151A) were consistent with the cis-proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.

Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody.

The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with significant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme specificity has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate specificity and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 A resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic efficiency and substrate specificity. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 A resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic specificity and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic efficiency incorporated in the procedure used to obtain it.

Engineering cyclophilin into a proline-specific endopeptidase.

Designing an enzyme requires, among a number of parameters, the appropriate positioning of catalytic machinery within a substrate-binding cleft. Using the structures of cyclophilin-peptide complexes, we have engineered a new catalytic activity into an Escherichia coli cyclophilin by mutating three amino acids, close to the peptide binding cleft, to form a catalytic triad similar to that found in serine proteases. In conjunction with cyclophilin's specificity for proline-bearing peptides, this creates a unique endopeptidase, cyproase 1, which cleaves peptides on the amino-side of proline residues. When acting on an Ala-Pro dipeptide, cyproase 1 has an efficiency (kcat/Km) of 0.7 x 10(4) M(-1) s(-1) and enhances the rate of reaction (kcat/kuncat) 8 x 10(8)-fold. This activity depends upon a deprotonated histidine and is inhibited by nucleophile-specific reagents, as occurs in natural serine proteases. Cyproase 1 can hydrolyse a protein substrate with a proline-specific endoprotease activity.

X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release.

The x-ray structures of the unliganded esterase-like catalytic antibody D2.3 and its complexes with a substrate analogue and with one of the reaction products are analyzed. Together with the structure of the phosphonate transition state analogue hapten complex, these crystal structures provide a complete description of the reaction pathway. At alkaline pH, D2.3 acts by preferential stabilization of the negatively charged oxyanion intermediate of the reaction that results from hydroxide attack on the substrate. A tyrosine residue plays a crucial role in catalysis: it activates the ester substrate and, together with an asparagine, it stabilizes the oxyanion intermediate. A canal allows facile diffusion of water molecules to the reaction center that is deeply buried in the structure. Residues bordering this canal provide targets for mutagenesis to introduce a general base in the vicinity of the reaction center.

Mechanism of inactivation of a catalytic antibody by p-nitrophenyl esters.

Antibody CNJ206 catalyses the hydrolysis of p-nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition-state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady-state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab-TSA X-ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.

Structural convergence in the active sites of a family of catalytic antibodies.

The x-ray structures of three esterase-like catalytic antibodies identified by screening for catalytic activity the entire hybridoma repertoire, elicited in response to a phosphonate transition state analog (TSA) hapten, were analyzed. The high resolution structures account for catalysis by transition state stabilization, and in all three antibodies a tyrosine residue participates in the oxyanion hole. Despite significant conformational differences in their combining sites, the three antibodies, which are the most efficient among those elicited, achieve catalysis in essentially the same mode, suggesting that evolution for binding to a single TSA followed by screening for catalysis lead to antibodies with structural convergence.

Similarities of hydrolytic antibodies revealed by their X-ray structures: a review.

Numerous antibodies have been programmed to catalyse the hydrolysis of esters as well as other acyl transfer reactions. They were raised against stable analogues that model the structure of the tetrahedral transition states of these reactions. The three-dimensional structures of four hydrolytic antibodies complexed to their respective phosphonate transition state analogues (TSAs) reveal a similar orientation of hapten relative to the antibody. Analysis of the four combining sites suggests that residues binding the phosphonate TSA stabilise the oxyanion intermediate of the reaction and play a preponderant role in catalysis. Comparison of catalytic antibodies selected from the same hybridoma fusion indicates a high similarity of the motifs that catalyse the hydrolysis of a given substrate.

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.

Aphakia correction by synthetic intracorneal implantation: a new tool for quantitative predetermination of the optical quality.

To improve the a priori estimation of optical quality of synthetic intracorneal implantation, numerical calculations were performed involving the intraocular overpressure, the cleavage depth and the type of the implant. Optical correction ratios, that were determined, are in close agreement with the obtained mechanical effects.