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-Å 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 ␤-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.

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.

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.

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