Simon Cocklin

The structure of the negative transcriptional regulator NmrA reveals a structural superfamily which includes the short‐chain dehydrogenase/reductases

NmrA is a negative transcriptional regulator involved in the post‐translational modulation of the GATA‐type transcription factor AreA, forming part of a system controlling nitrogen metabolite repression in various fungi. X‐ray structures of two NmrA crystal forms, both to 1.8 Å resolution, show NmrA consists of two domains, including a Rossmann fold. NmrA shows an unexpected similarity to the short‐chain dehydrogenase/reductase (SDR) family, with the closest relationship to UDP‐galactose 4‐epimerase. We show that NAD binds to NmrA, a previously unreported nucleotide binding property for this protein. NmrA is unlikely to be an active dehydrogenase, however, as the conserved catalytic tyrosine in SDRs is absent in NmrA, and thus the nucleotide binding to NmrA could have a regulatory function. Our results suggest that other transcription factors possess the SDR fold with functions including RNA binding. The SDR fold appears to have been adapted for other roles including non‐enzymatic control functions such as transcriptional regulation and is likely to be more widespread than previously recognized.

Enhanced EGFR inhibition and distinct epitope recognition by EGFR antagonistic mAbs C225 and 425.

Monoclonal antibodies (mAbs) that inhibit activation of the epidermal growth factor receptor (EGFR) have shown therapeutic potential in select malignancies including breast cancer. Here, we describe that combined use of two such mAbs, C225 (Cetuximab) and 425 (EMD55900), reduced growth and survival of EGFR overexpressing MDA-MB-468 breast cancer cells more effectively than either antibody alone. Similarly, the C225/425 antibody combination more effectively inhibited AKT and MAPK phosphorylation in MDA-MB-468 cells. Surface plasmon resonance, size exclusion chromatography, and analytical ultracentrifugation demonstrated that mAbs C225 and 425 simultaneously bind to distinct antigenic epitopes on domain III of the soluble wild-type EGFR. Furthermore, neither mAb competed with the other for binding to cells expressing either wild-type EGFR or a mutant EGFR (EGFRvIII) associated with neoplasia. Mutagenesis experiments revealed that residues S460/G461 in EGFR domain III are essential components of the 425 epitope and clearly distinguish it from the EGF/ TGF-α binding site and the C225 interaction interface. Collectively, these results support the conclusion that therapeutic EGFR blockade in cancer patients by combined use of mAbs C225 and 425 could provide advantages over the use of the two antibodies as single agents.

Inhibition of homologous recombination in human cells by targeting RAD51 recombinase.

The homologous recombination (HR) pathway plays a crucial role in the repair of DNA double-strand breaks (DSBs) and interstrand cross-links (ICLs). RAD51, a key protein of HR, possesses a unique activity: DNA strand exchange between homologous DNA sequences. Recently, using a high-throughput screening (HTS), we identified compound 1 (B02), which specifically inhibits the DNA strand exchange activity of human RAD51. Here, we analyzed the mechanism of inhibition and found that 1 disrupts RAD51 binding to DNA. We then examined the effect of 1 on HR and DNA repair in the cell. The results show that 1 inhibits HR and increases cell sensitivity to DNA damage. We propose to use 1 for analysis of cellular functions of RAD51. Because DSB- and ICL-inducing agents are commonly used in anticancer therapy, specific inhibitors of RAD51 may also help to increase killing of cancer cells.

Antibody Binding Is a Dominant Determinant of the Efficiency of Human Immunodeficiency Virus Type 1 Neutralization

ABSTRACT Primary and laboratory-adapted variants of human immunodeficiency virus type 1 (HIV-1) exhibit a wide range of sensitivities to neutralization by antibodies directed against the viral envelope glycoproteins. An antibody directed against an artificial FLAG epitope inserted into the envelope glycoproteins of three HIV-1 isolates with vastly different neutralization sensitivities inhibited all three viruses equivalently. Thus, naturally occurring HIV-1 isolates that are neutralization resistant are not necessarily more impervious to the inhibitory consequences of bound antibody. Moreover, the binding affinity of the anti-FLAG antibody correlated with neutralizing potency, underscoring the dominant impact on neutralization of antibody binding to the envelope glycoproteins.

PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific Protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA-)encoded peptide

The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E. coli protease Tsp has a single PDZ domain. PDZ domains are composed of short sequences (80–100 amino acids) that have been implicated in a range of protein:protein interactions. The PDZ-like domain of Tsp may be involved in binding to the extreme COOH-terminal sequence of its substrate, whereas the HtrA PDZ domains are involved in subunit assembly and are predicted to be responsible for substrate binding and subsequent translocation into the active site. E. coli has a system of protein quality control surveillance mediated by the ssrA-encoded peptide tagging system. This system tags misfolded proteins or protein fragments with an 11-amino acid peptide that is recognized by a battery of cytoplasmic and periplasmic proteases as a degradation signal. Here we show that both HtrA and Tsp are able to recognize the ssrA-encoded peptide tag with apparent K D values of ∼5 and 390 nm, respectively, and that their PDZ-like domains mediate this recognition. Fusion of the ssrA-encoded peptide tag to the COOH terminus of a heterologous protein (glutathioneS-transferase) renders it sensitive to digestion by Tsp but not HtrA. These observations support the prediction that the HtrA PDZ domains facilitate substrate binding and the differential proteolytic responses of HtrA and Tsp to SsrA-tagged glutathioneS-transferase are interpreted in terms of the structure of HtrA.

Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis

NmrA is a negative transcription‐regulating protein that binds to the C‐terminal region of the GATA transcription‐activating protein AreA. The proposed molecular mechanism of action for NmrA is to inhibit AreA binding to its target promoters. In contrast to this proposal, we report that a C‐terminal fragment of AreA can bind individually to GATA‐containing DNA and NmrA and that in the presence of a mixture of GATA‐containing DNA and NmrA, the AreA fragment binds preferentially to the GATA‐containing DNA in vitro. These observations are consistent with NmrA acting by an indirect route, such as by controlling entry into the nucleus. Deletion of the final nine amino acids of a C‐terminal fragment of AreA does not affect NmrA binding. Wild‐type NmrA binds NAD+(P+) with much greater affinity than NAD(P)H, despite the lack of the consensus GXXGXXG dinucleotide‐binding motif. However, introducing the GXXGXXG sequence into the NmrA double mutant N12G/A18G causes an ∼13‐fold increase in the KD for NAD+ and a 2.3‐fold increase for NADP+. An H37W mutant in NmrA designed to increase the interaction with the adenine ring of NAD+ has a decrease in KD of ∼4.5‐fold for NAD+ and a marginal 24% increase for NADP+. The crystal structure of the N12G/A18G mutant protein shows changes in main chain position as well as repositioning of H37, which disrupts contacts with the adenine ring of NAD+, changes which are predicted to reduce the binding affinity for this dinucleotide. The substitutions E193Q/D195N or Q202E/F204Y in the C‐terminal domain of NmrA reduced the affinity for a C‐terminal fragment of AreA, implying that this region of the protein interacts with AreA.

The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors

BackgroundHIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41s heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env.ResultsChimeric Env showed that the V1/V2-C2-V3 region in brains gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41s HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brains but not in spleens HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brains V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120s C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected.ConclusionWe have identified that the V1-V3 region of a brain-derived envelope glycoprotein seems to play a crucial role in determining not only the low CD4 dependence and increased macrophage tropism, but also the augmented fusogenicity and reduced sensitivity to T-1249 and BMS-378806. By contrast, increased sensitivity to HNG-105 mostly correlated with low CD4 dependence and macrophage tropism but was not determined by the presence of the brains V1-V3 region, confirming that viral determinants of phenotypic changes in brain-derived envelope glycoproteins are likely complex and context-dependent.

Structural Determinants for Affinity Enhancement of a Dual Antagonist Peptide Entry Inhibitor of Human Immunodeficiency Virus Type-1

Structure-activity correlations were investigated for substituted peptide conjugates that function as dual receptor site antagonists of HIV-1 gp120. A series of peptide conjugates were constructed via click reaction of both aryl and alkyl acetylenes with an internally incorporated azidoproline 6 derived from the parent peptide 1 (12p1, RINNIPWSEAMM). Compared to 1, many of these conjugates were found to exhibit several orders of magnitude increase in both affinity for HIV-1 gp120 and inhibition potencies at both the CD4 and coreceptor binding sites of gp120. We sought to determine structural factors in the added triazole grouping responsible for the increased binding affinity and antiviral activity of the dual inhibitor conjugates. We measured peptide conjugate potencies in both kinetic and cell infection assays. High affinity was sterically specific, being exhibited by the cis- but not the trans-triazole. The results demonstrate that aromatic, hydrophobic, and steric features in the residue 6 side-chain are important for increased affinity and inhibition. Optimizing these features provides a basis for developing gp120 dual inhibitors into peptidomimetic and increasingly smaller molecular weight entry antagonist leads.

Introducing metallocene into a triazole peptide conjugate reduces its off-rate and enhances its affinity and antiviral potency for HIV-1 gp120.

In this work, we identified a high affinity and potency metallocene‐containing triazole peptide conjugate that suppresses the interactions of HIV‐1 envelope gp120 at both its CD4 and co‐receptor binding sites. The ferrocene‐peptide conjugate, HNG‐156, was formed by an on‐resin copper‐catalysed [2 + 3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl‐containing triazole conjugate HNG‐105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV‐1 gp120 due mainly to the decreased dissociation rate of the conjugate‐gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037‐08) and clade B (YU‐2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV‐1YU‐2 gp120 at both the CD4 binding site and CD4‐induced binding sites. HNG‐156 showed a close‐to nanomolar IC50 for inhibiting cell infection by HIV‐1BaL whole virus. The dual receptor site antagonist activity and potency of HNG‐156 make it a promising viral envelope inhibitor lead for developing anti‐HIV‐1 treatments. Copyright

High-resolution crystal structure reveals molecular details of target recognition by bacitracin

Bacitracin is a metalloantibiotic agent that is widely used as a medicine and feed additive. It interferes with bacterial cell-wall biosynthesis by binding undecaprenyl-pyrophosphate, a lipid carrier that serves as a critical intermediate in cell wall production. Despite bacitracin’s broad use, the molecular details of its target recognition have not been elucidated. Here we report a crystal structure for the ternary complex of bacitracin A, zinc, and a geranyl-pyrophosphate ligand at a resolution of 1.1 Å. The antibiotic forms a compact structure that completely envelopes the ligand’s pyrophosphate group, together with flanking zinc and sodium ions. The complex adopts a highly amphipathic conformation that offers clues to antibiotic function in the context of bacterial membranes. Bacitracin’s efficient sequestration of its target represents a previously unseen mode for the recognition of lipid pyrophosphates, and suggests new directions for the design of next-generation antimicrobial agents.