Oligomeric states in sodium ion-dependent regulation of cyanobacterial histidine kinase-2

IMI thanks Queen Mary University of London for a graduate teaching studentship. LW thanks the China Scholarship Council (CSC) and Queen Mary University of London for financial support. SP held a Leverhulme Trust early-career post-doctoral research fellowship. JN is grateful for the continued support of the JST CREST Grant Number JPMJCR13M4, Japan. JFA acknowledges the support of research grant F/07 476/AQ and fellowship EM-2015-068 of the Leverhulme Trust

HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires \u3b22-microglobulin (\u3b22m) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface. In this study, we deepen the understanding of HLA-C and Env association by showing that HIV-1 specifically increases the amount of HLA-C free chains, not bound to \u3b22m, on the membrane of infected cells. The association between Env and HLA-C takes place at the cell membrane requiring \u3b22m to occur. We report that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C free chain molecules that have been correctly assembled with \u3b22m. HIV-1 Env-pseudotyped viruses produced in the absence of \u3b22m are less infectious than those produced in the presence of \u3b22m. We hypothesize that the conformation and surface expression of HLA-C molecules could be a discriminant for the association with Env. Binding stability to \u3b22m may confer to HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity

Chemical-genetic disruption of clathrin function spares adaptor complex 3-dependent endosome vesicle biogenesis

A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis

Does chemical cross-linking with NHS esters reflect the chemical equilibrium of protein-protein noncovalent interactions in solution?

Chemical cross-linking in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a powerful tool to study non-covalent protein complexes. Nevertheless, there are still many questions to answer: Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? In order to answer these questions, we performed systematic cross-linking studies with two complexes using the N8 hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): i) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a KD range of 30 nM to > 25 μM and ii) α-thrombin and basic pancreatic trypsin inhibitor (BPTI), which shows a buffer dependent KD value between 100 and 320 μM. Samples were analyzed by MALDI-MS. For NCoA-1•STAT6Y, a good correlation of the amount of cross-linked species with the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. The specificity of complex formation for the mid-affinity range up to about KD ≈ 25 μM could be proven by comparing against a non-binding peptide and by studying the concentration dependence. In order to study in which affinity range specific cross-linking can be applied, the weak α-thrombin•BPTI complex was investigated. Although variations of the sodium concentration can change the dissociation constant up to 3-fold for this interaction, no significant effect on the amount of detected complex was observed at different peptide concentrations. Our interpretation of this result is that the detected complex is not specific, but a nonspecifically cross-linked species. Consequently, chemical cross-linking is not applicable to low-affinity complexes with KD >> 25 μM with the experimental approach used in this study