The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Crystal structures have shown that the HIV-1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi-open are observed in the free protease, crystal structures reveal that even the semi-open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub-ns) dynamics of the flaps of the free protease in solution, to relate these results to X-ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free-protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of 3h J NCЈ couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49-53. Model-free analysis of 15 N relaxation parameters, T 1 , T 2 (T 1 ) and 15 N-{ 1 H} NOE, shows that residues in the flap tips are flexible on the sub-ns time scale, in contrast with previous observations on the inhibitor-bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub-ns time scale dynamics of the flap tips is discussed.

Sgt1 has been identified as a subunit of both core kinetochore and SCF (Skp1-Cul1-F-box) ubiquitin ligase complexes and is also implicated in plant disease resistance. Sgt1 has two putative HSP90 binding domains, a tetratricopeptide repeat and a p23-like CHORD and Sgt1 (CS) domain. Using NMR spectroscopy, we show that only the CS domain of human Sgt1 physically interacts with HSP90. The tetratricopeptide repeat domain does not bind to either HSP90 or HSP70. Determination of the three-dimensional structure showed that the Sgt1-CS domain shares the same ␤-sandwich fold as p23 but lacks the last highly conserved ␤-strand in p23. Analysis of the structures of Sgt1-CS and p23 revealed a similar charge distribution on one of two opposing surfaces that suggests that it is the binding region for HSP90 in Sgt1. Although ATP is absolutely required for p23 binding to HSP90, Sgt1 binds to HSP90 also in the absence of the non-hydrolyzable analog ATP␥S. Our findings suggest the CS domain is a binding module for HSP90 distinct from p23-like domains, which implies that Sgt1 and related proteins function in recruiting heat shock protein activities to multiprotein assemblies. Heat shock protein 90 (HSP90)1 is a molecular chaperone important for protein folding. HSP90 is different from other chaperones because most of its substrates are related to signal transduction (1). Recent studies also suggest that HSP90 plays a role in protein quality control where it facilitates the polyubiquitination and degradation of substrates through interaction with the co-chaperone C terminus of HSC70-interacting protein (CHIP) (2). Thus, HSP90 can be involved in protein regulation in quite different ways depending on the cellular context.Very recently, it has been reported that Sgt1 interacts with HSP90 (3-5). Sgt1 was originally identified as a suppressor of the G 2 allele of Skp1 and was found to be important for both the G 1 /S and G 2 /M transitions in the cell cycle (6). The G 2 /M transition involves activation of the kinetochore. Sgt1 was shown to be required for the activation of the kinetochore core complex CBF3 and to physically interact with Skp1, one component of CBF3 (6). Sgt1 also physically interacts with Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes through interaction with Skp1. Moreover, a yeast Sgt1 mutant was defective in Sic1 degradation through ubiquitination (6).Sequence analysis of Sgt1 proteins from yeast, human, barley, rice, and Arabidopsis shows three conserved domains (tetratricopeptide repeat (TPR), CHORD-containing proteins and Sgt1 (CS), and Sgt1-specific (SGS)) and two variable regions (VR1 and VR2) (7). TPR domains are known as heat shock protein binding domains. The SGS domain was shown to interact with S100 calcium-binding proteins (8). The CS domain has high sequence homology with p23 and is also known as a p23-like domain (9). p23 has been shown to physically and functionally interact with HSP90, apparently serving in a role as co-chaperone. Recently, HSP90 was shown to be an essential factor required for...

The crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts were monitored in single-stranded and duplex oligodeoxynucleotides using NMR spectroscopy. In both instances, the cis and trans diastereomers of the alpha-CH3 and gamma-OH groups underwent slow exchange, with the trans diastereomers being favored. In single-stranded oligodeoxynucleotides, the aldehyde intermediates were not detected spectroscopically, but their presence was revealed through the formation of N-terminal conjugates with the tetrapeptide KWKK. When annealed into 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCYCTAGC)-3' containing the 5'-CpG-3' sequence context (X = R- or S-alpha-CH3-gamma-13C-OH-PdG; Y = 15N2-dG) at pH 7, partial opening of the R- or S-alpha-CH3-gamma-13C-OH-PdG adducts to the corresponding N2-(3-oxo-1-methyl-propyl)-dG aldehydes was observed at temperatures below the T(m) of the duplexes. These aldehydes equilibrated with their geminal diol hydrates; higher temperatures favored the aldehydes. When annealed opposite T, the S-alpha-CH3-gamma-13C-OH-PdG adduct was stable. At 37 degrees C, an interstrand DNA cross-link was observed spectroscopically only for the R-alpha-CH3-gamma-OH-PdG adduct. Molecular modeling predicted that the interstrand cross-link formed by the R-alpha-CH3-gamma-OH-PdG adduct introduced less disruption into the duplex structure than did the cross-link arising from the S-alpha-CH3-gamma-OH-PdG adduct, due to differing orientations of the R- and S-CH3 groups. Modeling also predicted that the alpha-methyl group of the aldehyde arising from the R-alpha-CH3-gamma-OH-PdG adduct is oriented in the 3'-direction in the minor groove, facilitating cross-linking. In contrast, the alpha-methyl group of the aldehyde arising from the S-alpha-CH3-gamma-OH-PdG adduct is oriented in the 5'-direction within the minor groove, potentially hindering cross-linking. NMR revealed that for the R-alpha-CH3-gamma-OH-PdG adduct, the carbinolamine form of the cross-link was favored in duplex DNA with the imine (Schiff base) form of the cross-link remaining below the level of spectroscopic detection. Molecular modeling predicted that the carbinolamine linkage maintained Watson-Crick hydrogen bonding at both of the tandem C.G base pairs. Dehydration of the carbinolamine cross-link to an imine, or cyclization of the latter to form a pyrimidopurinone cross-link, required disruption of Watson-Crick hydrogen bonding at one or both of the cross-linked base pairs.

Backbone nuclear magnetic resonance (NMR) assignments were achieved for diacylglycerol kinase (DAGK) in detergent micelles. DAGK is a homotrimeric integral membrane protein comprised of 121 residue subunits, each having three transmembrane segments. Assignments were made using TROSY-based pulse sequences. DAGK was found to be an almost exclusively helical protein. This work points to the feasibility of both solving the structure of DAGK using solution NMR methods and using NMR as a primary tool in structural studies of other helical integral membrane proteins of similar size and complexity.

The interstrand N 2 ,N 2 -dG DNA crosslinking chemistry of the acrolein-derived γ-OH-1,N 2 -propanodeoxyguanosine (γ-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13 C-and 15 N-edited experiments. At equilibrium 40% of the DNA was crosslinked, with the carbinolamine form of the crosslink predominating. The crosslink existed in equilibrium with the non-crosslinked N 2 -(3-oxopropyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde:diol increased at higher temperatures. The 1,N 2 -dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C•G base pairs. In contrast, dehydration of the carbinolamine crosslink to an imine (Schiff base) crosslink, or cyclization of the latter to form a pyrimidopurinone crosslink, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem crosslinked C•G base pairs. When the γ-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N 2 -dG cyclic adduct, the aldehyde, and the diol, but no crosslink, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide crosslinks efficiently. When annealed opposite dA, γ-OH-PdG remained as the 1,N 2 -dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand crosslinks formed by acrolein and perhaps other α,β-unsaturated aldehydes. These sequence-specific carbinolamine crosslinks are anticipated to interfere with DNA replication and contribute to acroleinmediated genotoxicity.

Alamethicin is a helical 20-amino acid voltage-gated channel-forming peptide, which is known to exhibit segmental flexibility in solution along its backbone near alpha-methylalanine (MeA)-10 and Gly-11. In an alpha-helical configuration, MeA at position 10 would normally hydrogen-bond with position 14, but the presence of proline at this position prevents the formation of this interhelical hydrogen bond. To determine whether the presence of proline at position 14 contributes to the flexibility of this helix, two analogs of alamethicin were synthesized, one with proline 14 replaced by alanine and another with both proline 14 and glycine 11 replaced by alanine. The C-termini of these peptides were derivatized with a proxyl nitroxide, and paramagnetic enhancements produced by the nitroxide on the Calpha protons were used to estimate r-6 weighted distances between the nitroxide and the backbone protons. When compared to native alamethicin, the analog lacking proline 14 exhibited similar C-terminal to Calpha proton distances, indicating that substitution of proline alone does not alter the flexibility of this helix; however, the subsequent removal of glycine 11 resulted in a significant increase in the averaged distances between the C- and N-termini. Thus, the G-X-X-P motif found in alamethicin appears to be largely responsible for mediating high-amplitude bending motions that have been observed in the central helical domain of alamethicin in methanol. To determine whether these substitutions alter the channel behavior of alamethicin, the macroscopic and single-channel currents produced by these analogs were compared. Although the substitution of the G-X-X-P motif produces channels with altered characteristics, this motif is not essential to achieve voltage-dependent gating or alamethicin-like behavior.

LP2086 is a family of outer membrane lipoproteins fromNeisseria meningitidis, which elicits bactericidal antibodies and are currently undergoing human clinical trials in a bivalent formulation where each antigen represents one of the two known LP2086 subfamilies. Here we report the NMR structure of the recombinant LP2086 variant B01, a representative of the LP2086 subfamily B. The structure reveals a novel fold composed of two domains: a "taco-shaped" N-terminal ␤-sheet and a C-terminal ␤-barrel connected by a linker. The structure in micellar solution is consistent with a model of LP2086 anchored to the outer membrane bilayer through its lipidated N terminus. A long flexible chain connects the folded part of the protein to the lipid anchor and acts as spacer, making both domains accessible to the host immune system. Antibodies broadly reactive against members from both subfamilies have been mapped to the N terminus. A surface of subfamily-defining residues was identified on one face of the protein, offering an explanation for the induction of subfamily-specific bactericidal antibodies.Neisseria meningitidis is a Gram-negative bacterial pathogen, which colonizes the upper respiratory tract, occasionally invading the bloodstream, causing sepsis, and crossing the blood-brain barrier, resulting in meningitis. Despite the availability of effective antibiotic treatment, the rapid progression of meningococcal disease still results in substantial morbidity and mortality (1). Five meningococcal serogroups, categorized according to the chemical structure of the bacterial capsular polysaccharides, A, B, C, Y, and W135, account for most of the disease (2). Although a vaccine against four of the five major serogroups of meningococci is currently available, a vaccine for the prevention of serogroup B disease is still an unmet clinical need (3). The development of vaccines against serogroup B meningococci has focused on subcapsular antigens, in order to avoid the risk of autoimmunity arising from structural similarities between the capsular polysaccharides and the sialic acidmodified surface of developing human brain (1,4,5).Recently, a new family of lipidated outer membrane proteins, LP2086, was identified as a potential vaccine target (6). Members of the LP2086 family have been divided into two subfamilies, subfamily A and B, based on their genetic variation (6, 7). Since recombinant LP2086 (rLP2086) 3 elicits a bactericidal response that is largely subfamily-specific, a bivalent vaccine containing one protein from each subfamily will offer protection against serogroup B meningococci (6, 8 -11). LP2086 lipoproteins are lipidated at the N-terminal Cys with a tripalmitoyl lipid tail, which anchors the protein to the bacterial membrane (12). More recently, LP2086 was found to induce serum resistance via binding with human Factor H, a key regulator of the alternative complement pathway that prevents autologous complement attack (13).Our work seeks to understand the structural elements of LP2086 responsible for inducing the subf...