Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor.

WW domains are small β-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.

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.

In infected cells the RNA genome of FMDV is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally-encoded proteases. Most of these cleavages are performed by the highly-conserved 3C protease (3Cpro), making the enzyme an attractive target for antiviral drugs. Such drugs could provide a valuable prophylactic weapon during FMD epidemics in previously disease-free regions by reducing transmission in the period before vaccination takes effect.

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 β-barrel of the second repeat.

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.

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, ɛ and γ. 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.

Engineered cyclotides. The exceptionally potent trypsin inhibitor macrocyclic cysteine-knot microproteins (or "cyclotides") MCoTI-I and MCoTI-II and analogues were synthesised by one-pot protease-mediated backbone ligation/affinity purification and/or thia-zip native chemical ligation cascade cyclization. One of these MCoTI analogues (MCoTI-II[K10F]) is the first example of an engineered MCoTI cyclotide in which specificity is redirected towards an alternative enzyme target (chymotrypsin), whilst retaining high affinity. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2007/z700187_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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, 3Cpro, 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 kcat/KM of 57.6 ± 2.0 M−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 3Cpro.

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 β-hairpin structures.

Proteinaceous proteinase inhibitors control proteases’ proteolytic activity by preventing unwanted proteolysis. They are involved in important biological “housekeeping” functions such as blood clotting, inflammatory reactions etc. The BBI family are small serine protease inhibitors (60 to 90 amino acids) which are particularly abundant in leguminous and gramineous plants [1,2]. They contain 7 disulfide bridges and have a symmetrical double-headed structure. Each domain contains an independent canonical binding site of nine amino acid residues encapsulated in a disulfide bridge. Generally, the inhibitory activity is directed at either chymotrypsin/trypsin or trypsin/trypsin.