John E. Ladbury

A Landscape of Driver Mutations in Melanoma

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19-harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.

Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.

Hsp90 molecular chaperones in eukaryotic cells play essential roles in the folding and activation of a range of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction. The biochemical mechanism of Hsp90 is poorly understood, and the involvement of ATP in particular is controversial. Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific adenine nucleotide binding site homologous to the ATP-binding site of DNA gyrase B. This site is the same as that identified for the antitumor agent geldanamycin, suggesting that geldanamycin acts by blocking the binding of nucleotides to Hsp90 and not the binding of incompletely folded client polypeptides as previously suggested. These results finally resolve the question of the direct involvement of ATP in Hsp90 function.

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90‐specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP‐coupled chaperone cycle for Hsp90‐mediated protein folding.

Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation

Heparin is required for fibroblast growth factor (FGF) stimulation of biological responses. Using isothermal titration calorimetry, we show that acidic FGF (aFGF) forms a 1:1 complex with the soluble extracellular domain of FGF receptor (FGFR). Heparin exerts its effect by binding to many molecules of aFGF. The resulting aFGF-heparin complex can bind to several receptor molecules, leading to FGFR dimerization. In two cell lines lacking endogenous heparan sulfate, exogenous heparin is required for FGFR dimerization, tyrosine kinase activation, c-fos mRNA transcription, and cell proliferation. Moreover, a synthetic heparin analog that binds monovalently to aFGF blocks FGFR dimerization, activation, and signaling via FGFR. We propose that heparin causes oligomerization of aFGF such that its binding to FGFR results in dimerization and activation. This represents a novel mechanism for transmembrane signaling and may account for the action of many heparin-bound growth factors.

The ATPase cycle of Hsp90 drives a molecular ‘clamp’ via transient dimerization of the N-terminal domains

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C‐terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5′‐adenylamido‐diphosphate (AMP‐PNP), and AMP‐PNP‐ promoted association of N‐termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C‐terminal truncation mutants also required AMP‐PNP‐dependent dimerization. The temperature‐ sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP‐PNP‐dependent N‐terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP‐PNP‐dependent N‐terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the ‘lid’ segment (residues 100–121) couples ATP binding to N‐terminal association. Consistent with this, a mutation designed to favour ‘lid’ closure (A107N) substantially enhanced ATPase activity and N‐terminal dimerization. These data show that Hsp90 has a molecular ‘clamp’ mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N‐terminal dimerization are directly coupled to the ATPase cycle.

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)- domain co-chaperones

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co‐chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR‐domain co‐chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub‐micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co‐chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP‐binding site in the N‐terminal domain of Hsp90. These results reveal an important role for TPR‐domain co‐chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP‐dependent step in Hsp90‐mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client‐protein release.

Two EGF molecules contribute additively to stabilization of the EGFR dimer

Receptor dimerization is generally considered to be the primary signaling event upon binding of a growth factor to its receptor at the cell surface. Little, however, is known about the precise molecular details of ligand‐induced receptor dimerization, except for studies of the human growth hormone (hGH) receptor. We have analyzed the binding of epidermal growth factor (EGF) to the extracellular domain of its receptor (sEGFR) using titration calorimetry, and the resulting dimerization of sEGFR using small‐angle X‐ray scattering. EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data obtained from the two approaches suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF‐sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor‐receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF‐induced sEGFR dimerization from the hGH and interferon‐γ receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF‐induced sEGFR dimerization suggests possible mechanisms for both ligand‐induced homo‐ and heterodimerization of the EGFR (or erbB) family of receptors.

Just add water! The effect of water on the specificity of protein-ligand binding sites and its potential application to drug design

Recent data have highlighted the enigmatic role that water plays in biomolecular complexes. Water at the interface of a complex can increase the promiscuity of an interaction, yet it can also provide increased specificity and affinity. The ability to engineer water-binding sites into the interface between a drug and its target might prove useful in drug design.

TCR Binding to Peptide-MHC Stabilizes a Flexible Recognition Interface

The binding of TCRs to their peptide-MHC ligands is characterized by a low affinity, slow kinetics, and a high degree of cross-reactivity. Here, we report the results of a kinetic and thermodynamic analysis of two TCRs binding to their peptide-MHC ligands, which reveal two striking features. First, significant activation energy barriers must be overcome during both association and dissociation, suggesting that conformational adjustments are required. Second, the low affinity of binding is a consequence of highly unfavorable entropic effects, indicative of a substantial reduction in disorder upon binding. This is evidence that the TCR and/or peptide-MHC have flexible binding surfaces that are stabilized upon binding. Such conformational flexibility, which may also be a feature of primary antibodies, is likely to contribute to cross-reactivity in antigen recognition.

Adding calorimetric data to decision making in lead discovery: a hot tip

Recognition of the limitations of high-throughput screening approaches in the discovery of candidate drugs has reawakened interest in structure-based and other rational design methods. Here, we describe how isothermal titration calorimetry can be used to obtain thermodynamic data on the binding of compounds to protein targets. We propose that these data — particularly the change in enthalpy — could provide a valuable, complementary addition to established tools for selecting compounds in lead discovery and for aiding lead optimization.