Agnès M. Jaulent

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, 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.

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.

Immobilized Protease‐Assisted Synthesis of Engineered Cysteine‐Knot Microproteins

Cyclotides are a unique class of head-to-tail, cyclic, cysteinerich microproteins up to 37 amino acids in length that exhibit a wide range of biological properties, ranging from anti-HIV to insecticidal activity. In contrast to many smaller naturally occurring cyclic peptides, they are true gene products, and possess a highly stable cysteine-knot topology whereby two disulfide bridges form a ring through which a third is threaded. This combination of intricate structure, diverse biological activity and unusual biogenesis has inspired a growing research effort directed towards understanding the synthesis and function of cyclotides both in the cell and in vitro. In addition, their high resistance to degradation in vivo has attracted recent interest in the potential of cyclotides as scaffolds to present structured protein domains for therapeutic applications. We recently reported the first total synthesis of MCoTI-II, the founding member of a small cyclotide family that possesses exceptionally potent trypsin-inhibitory activity (Ki<100 pm). MCoTI-II was first isolated from Momordica cochinchinensis, and its solution structure as determined by NMR is shown in Figure 1. Cyclotide total synthesis presents three major challenges: 1) synthesis of the peptide backbone, which in MCoTI cycloACHTUNGTRENNUNGtides contains a sensitive Asp-Gly (DG) motif ; 2) controlled head-to-tail cyclization to form a very large macrolactam (102 atoms in the case of MCoTI cyclotides) ; and 3) folding into the native tetracyclic knot structure. Our synthesis of MCoTI-II, like other successful cyclotide syntheses, made use of thia-zip native chemical ligation (NCL) cyclization, a technique pioneered by the Tam group whereby an open-chain backbone bearing a C-terminal thioester and an N-terminal cysteine progresses through a series of intermediate cyclic thioesters, the largest of which is trapped by NCL to give the target macrolactam. However, this elegant approach requires the often nontrivial synthesis of a large peptide thioester and multiple purification steps that can significantly reduce the potential overall yield. Recent reports have demonstrated the potential of certain proteases to act as peptide ligases under carefully optimized conditions. We considered that the presence of a well-defined trypsin binding site in MCoTI peptides coupled with the reversibility displayed in protease cleavage of disulfide-bonded head-to-tail cyclic peptides might permit protease-mediated ligation of the cyclotide backbone between positions P1 and P1’ in the active loop (i.e. , Lys10-Ile11) in a manner analogous to that of the noncyclotide trypsin inhibitors SFTI-I and BPTI. The requisite Ile11-Lys10 C-terminal acid peptide backbone starting material (oc-MCoTI-II) was synthesised in excellent overall yield by using the Fmoc/tBu solid-phase peptide synthsis (SPPS) protocol. As in our previous synthesis of MCoTI-II, aspartimide formation at the DG motif was effectively suppressed by the introduction of 2-hydroxy-4-methoxybenzyl (Hmb) protection at the glycine backbone nitrogen. ocMCoTI-II was refolded to cysteine-knot peptide rf-MCoTI-II by exposure to a mild oxidant (reduced glutathione); under optimized conditions (Scheme 1) no misfolded peptide was observed by HPLC. In initial attempts to achieve backbone ligation, mixtures of rf-MCoTI-II and trypsin (up to 1:1 ratio) were combined in 100 mm phosphate buffer (pH 7.4). In each case, some cyclization to MCoTI-II was seen by MALDI and HPLC (by comparison with authentic MCoTI-II prepared by one-pot thia-zip chemistry), but contamination with peptides resulting from protease autodigestion precluded further analysis and purification. Trypsin immobilized on Sepharose beads (polymer-supported trypsin, PST) is used widely for sequencing applications, since contamination through autocleavage is greatly suppressed, and we considered that we might be able to employ PST as a polymer-supported ligase for clean MCoTI macrolactamization. In a typical binding procedure (Scheme 1) a two[a] P. Thongyoo, Dr. E. W. Tate, Prof. R. J. Leatherbarrow Department of Chemistry, Imperial College London Exhibition Road, London SW7 2AZ (UK) Fax: (+44) 2075941139 E-mail : e.tate@imperial.ac.uk r.j.leatherbarrow@imperial.ac.uk [b] Dr. A. M. Jaulent Current address: MRC Laboratory of Molecular Biology Hills Road, Cambridge, CB2 0QH (UK) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Figure 1. Sequence and solution structure of MCoTI-II. 9] The side chains of Pro9, Lys10 (P1) and Ile11 (P1’) and disulfide bonds are shown as sticks. Presented with PyMol.

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.

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.

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 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.

Synthesis and Activity of a Small Cyclic Protease Inhibitor from Sunflower Seeds, SFTI-1

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.