Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display.

WW domains are small ß-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications.

Posttranslational modifications affect the interaction of S100 proteins with tumor suppressor p53.

Proteins of the S100 family bind to the intrinsically disordered transactivation domain (TAD; residues 1-57) and C-terminus (residues 293-393) of the tumor suppressor p53. Both regions provide sites that are subject to posttranslational modifications, such as phosphorylation and acetylation, that can alter the affinity for interacting proteins such as p300 and MDM2. Here, we found that S100A1, S100A2, S100A4, S100A6, and S100B bound to two subdomains of the TAD (TAD1 and TAD2). Both subdomains were mandatory for high-affinity binding to S100 proteins. Phosphorylation of Ser and Thr residues increased the affinity for the p53 TAD. Conversely, acetylation and phosphorylation of the C-terminus of p53 decreased the affinity for S100A2 and S100B. In contrast, we found that nitrosylation of S100B caused a minor increase in binding to the p53 C-terminus, whereas binding to the TAD remained unaffected. As activation of p53 is usually accompanied by phosphorylation and acetylation at several sites, our results suggest that a shift in binding from the C-terminus in favor of the N-terminus occurs upon the modification of p53. We propose that binding to the p53 TAD might be involved in the stimulation of p53 activity by S100 proteins.

Moving towards high-resolution descriptions of the molecular interactions and structural rearrangements of the human hepatitis B core protein.

The human hepatitis B virus core protein (HBc) forms icosahedral capsids and plays central roles in viral replication. The critical interactions that HBc makes prior to capsid formation (potential drug targets) have proved refractory to structural characterisation as HBc aggressively forms capsids. Our current structural understanding of HBc interactions is therefore capsid-centric, and this view has been limited by the resolution of cryo-electron microscopy and the inherent difficulties in getting high-quality crystals of viral capsids. To augment these approaches, we used capsid-dissociating conditions, solution NMR, and biophysical methodologies to directly characterise, at atomic resolution, the structural properties of dimeric HBc, as well as its dynamics and intermolecular interactions. Dimeric HBc recapitulates the structural properties and binding interactions of HBc within the context of capsids. Antiviral peptides induced long-range structural asymmetry in dimeric HBc, providing new insights into how ligand binding can effect communication between different regions of HBc and, therefore, between the capsid interior and the capsid exterior. Our work also paves the way for detailed descriptions of the previously invisible early stages of replication involving soluble HBc.

14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers.

Activation of the tumour suppressor p53 on DNA damage involves post-translational modification by phosphorylation and acetylation. Phosphorylation of certain residues is critical for p53 stabilization and plays an important role in DNA-binding activity. The 14-3-3 family of proteins activates the DNA-binding affinity of p53 upon stress by binding to a site in its intrinsically disordered C-terminal domain containing a phosphorylated serine at 378. We have screened various p53 C-terminal phosphorylated peptides for binding to two different isoforms of 14-3-3, epsilon and gamma. We found that phosphorylation at either S366 or T387 caused even tighter binding to 14-3-3. We made by semi-synthesis a tetrameric construct comprised of the tetramerization plus C-terminal domains of p53 that was phosphorylated on S366, S378 and T387. It bound 10 times tighter than did the monomeric counterpart to dimeric 14-3-3. We showed indirectly from binding curves and directly from fluorescence-detection analytical ultracentrifugation that 14-3-3 enhanced the binding of sequence-specific DNA to p53 by causing p53 dimers to form tetramers at lower concentrations. If the in vitro data extrapolate to in vivo, then it is an attractive hypothesis that p53 activity may be subject to control by accessory proteins lowering its tetramer-dimer dissociation constant from its normal value of 120-150 nM.

The malignant brain tumor repeats of human SCML2 bind to peptides containing monomethylated lysine.

SCML2 (sex comb on midleg-like 2) is a constituent of the Polycomb repressive complex 1, a large multiprotein assembly required for the repression of developmental control genes. It contains two MBT (malignant brain tumor) repeats; the MBT is a protein module structurally similar to domains that bind to methylated histones. We have used NMR spectroscopy to examine the binding specificity of these repeats. Our data show that they preferentially bind histone peptides monomethylated at lysine residues with no apparent sequence specificity. The crystal structure of the complex between the protein and monomethyllysine reveals that the modified amino acid binds to an aromatic rich pocket at one end of the beta-barrel of the second repeat.

A continuous assay for foot-and-mouth disease virus 3C protease activity.

Foot-and-mouth disease virus is a highly contagious pathogen that spreads rapidly among livestock and is capable of causing widespread agricultural and economic devastation. The virus genome is translated to produce a single polypeptide chain that subsequently is cleaved by viral proteases into mature protein products, with one protease, 3C(pro), carrying out the majority of the cleavages. The highly conserved nature of this protease across different viral strains and its crucial role in viral maturation and replication make it a very desirable target for inhibitor design. However, the lack of a convenient and high-throughput assay has been a hindrance in the characterization of potential inhibitors. In this article, we report the development of a continuous assay with potential for high throughput using fluorescence resonance energy transfer-based peptide substrates. Several peptide substrates containing the 3C-specific cleavage site were synthesized, varying both the positions and separation of the fluorescent donor and quencher groups. The best substrate, with a specificity constant k(cat)/K(M) of 57.6+/-2.0M(-1) s(-1), was used in inhibition assays to further characterize the protease's activity against a range of commercially available inhibitors. The inhibition profile of the enzyme showed characteristics of both cysteine and serine proteases, with the chymotrypsin inhibitor TPCK giving stoichiometric inhibition of the enzyme and allowing active site titration of the 3C(pro).

Solution structure of a novel C2-symmetrical bifunctional bicyclic inhibitor based on SFTI-1.

A novel bifunctional bicyclic inhibitor has been created that combines features both from the Bowman-Birk inhibitor (BBI) proteins, which have two distinct inhibitory sites, and from sunflower trypsin inhibitor-1 (SFTI-1), which has a compact bicyclic structure. The inhibitor was designed by fusing together a pair of reactive loops based on a sequence derived from SFTI-1 to create a backbone-cyclized disulfide-bridged 16-mer peptide. This peptide has two symmetrically spaced trypsin binding sites. Its synthesis and biological activity have been reported in a previous communication [Jaulent and Leatherbarrow, 2004, PEDS 17, 681]. In the present study we have examined the three-dimensional structure of the molecule. We find that the new inhibitor, which has a symmetrical 8-mer half-cystine CTKSIPP'I' motif repeated through a C2 symmetry axis also shows a complete symmetry in its three-dimensional structure. Each of the two loops adopts the expected canonical conformation common to all BBIs as well as SFTI-1. We also find that the inhibitor displays a strong and unique structural identity, with a notable lack of minor conformational isomers that characterise most reactive site loop mimics examined to date as well as SFTI-1. This suggests that the presence of the additional cyclic loop acts to restrict conformational mobility and that the deliberate introduction of cyclic symmetry may offer a general route to locking the conformation of beta-hairpin structures.

Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity.

Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C(pro)), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C(pro), determined the crystal structure to 1.9-angstroms resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C(pro) adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4-P4') and that FMDV 3C(pro) discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C(pro) enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C(pro) of an extended beta-ribbon that folds over the substrate binding cleft in other picornavirus 3C(pro) structures. Collectively, these results establish a valuable framework for the development of FMDV 3C(pro) inhibitors.

Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors.

Two novel synthetic inhibitors were designed to combine the advantageous properties of Bowman Birk inhibitor (BBI) and sunflower trypsin inhibitor-1 (SFTI-1). As is the case for BBI, the novel inhibitors have two active sites that give dual independent protease inhibition. However, they also possess a small bicyclic structure, reminiscent of the single-site SFTI-1. It is found that the synthetic inhibitors retain the potent inhibitory properties of the parent structures; they are also found to be relatively resistant to proteolysis. Their inhibition properties and a comparison of their stability to proteolysis relative to SFTI-1 are described. It is found that the new inhibitors do indeed allow bifunctional inhibition, although, unlike BBI, the small size of the inhibitor prevents the simultaneous inhibition of two proteases at the same time.

Peptide mimics of the Bowman-Birk inhibitor reactive site loop.

Bowman-Birk Inhibitors (BBIs) are small highly cross-linked proteins that typically display an almost symmetrical "double-headed" structure. Each "head" contains an independent proteinase binding domain. The realization that one BBI molecule could form a 1:1:1 complex with two enzymes led early workers to dissect this activity. Now, after three decades of research, it has been possible to isolate the antiproteinase activity as small ( approximately 11 residues), cyclic, synthetic peptides, which display most of the functional aspects of the protein. More recently, it has been found that these peptide fragments are not just a synthetic curiosity-a natural 14-residue cyclic peptide (SFTI-1), which too encapsulates the BBI inhibitory motif, is found to occur in sunflowers. This article reviews the properties of BBI-based peptides (including SFTI-1) and discusses the features that are important for inhibitory activity.