Detection and Architecture of Small Heat Shock Protein Monomers

International audienceBACKGROUND: Small Heat Shock Proteins (sHSPs) are chaperone-like proteins involved in the prevention of the irreversible aggregation of misfolded proteins. Although many studies have already been conducted on sHSPs, the molecular mechanisms and structural properties of these proteins remain unclear. Here, we propose a better understanding of the architecture, organization and properties of the sHSP family through structural and functional annotations. We focused on the Alpha Crystallin Domain (ACD), a sandwich fold that is the hallmark of the sHSP family. METHODOLOGY/PRINCIPAL FINDINGS: We developed a new approach for detecting sHSPs and delineating ACDs based on an iterative Hidden Markov Model algorithm using a multiple alignment profile generated from structural data on ACD. Using this procedure on the UniProt databank, we found 4478 sequences identified as sHSPs, showing a very good coverage with the corresponding PROSITE and Pfam profiles. ACD was then delimited and structurally annotated. We showed that taxonomic-based groups of sHSPs (animals, plants, bacteria) have unique features regarding the length of their ACD and, more specifically, the length of a large loop within ACD. We detailed highly conserved residues and patterns specific to the whole family or to some groups of sHSPs. For 96% of studied sHSPs, we identified in the C-terminal region a conserved I/V/L-X-I/V/L motif that acts as an anchor in the oligomerization process. The fragment defined from the end of ACD to the end of this motif has a mean length of 14 residues and was named the C-terminal Anchoring Module (CAM). CONCLUSIONS/SIGNIFICANCE: This work annotates structural components of ACD and quantifies properties of several thousand sHSPs. It gives a more accurate overview of the architecture of sHSP monomers

Targeting cysteine thiols for in vitro site-specific glycosylation of recombinant proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature

The Liganding of Glycolipid Transfer Protein Is Controlled by Glycolipid Acyl Structure

Glycosphingolipids (GSLs) play major roles in cellular growth and development. Mammalian glycolipid transfer proteins (GLTPs) are potential regulators of cell processes mediated by GSLs and display a unique architecture among lipid binding/transfer proteins. The GLTP fold represents a novel membrane targeting/interaction domain among peripheral proteins. Here we report crystal structures of human GLTP bound to GSLs of diverse acyl chain length, unsaturation, and sugar composition. Structural comparisons show a highly conserved anchoring of galactosyl- and lactosyl-amide headgroups by the GLTP recognition center. By contrast, acyl chain chemical structure and occupancy of the hydrophobic tunnel dictate partitioning between sphingosine-in and newly-observed sphingosine-out ligand-binding modes. The structural insights, combined with computed interaction propensity distributions, suggest a concerted sequence of events mediated by GLTP conformational changes during GSL transfer to and/or from membranes, as well as during GSL presentation and/or transfer to other proteins

DNA Binding Specificity of Mu Transcription Factor C and Crystallization of C : DNA Complex

The lytic cycle of phage Mu is regulated by a transcriptional cascade consisting of early, middle and late transcription. The Mor protein is an activator of the middle promoter Pm and is encoded by the last gene of the early transcript. The C protein is an activator of the four late promoters Plys, PI, PP, and Pmom and is expressed from the middle transcript. Both Mor and C proteins bind an imperfect dyad-symmetry element just upstream and overlapping the –35 region of Pm and Plys respectively. The main aims of this study was, (1) To understand the binding specificity of C and determine a possible consensus sequence for C binding, and (2) To crystallize the C : DNA complex as a first step towards structure determination. In previous work, single base substitution mutations in Plys identified bases and positions important for C binding and activation. To get a consensus sequence for C binding, we tested additional candidate mutations within and flanking the C binding sequence. Wild-type C protein was used in gel mobility shift assays with annealed oligonucleotides containing mutations, insertions and deletions. The assay showed that, (1) mutation in positions –53, –52 and –32 did not affect C binding, (2) mutations flanking the IR spacer (–40, –41, –46, –47) influence C binding, and (3) insertion or deletion of a single base pair in the IR spacer abolished C binding. Mor and C proteins are the founding members of a new class of transcription factors. The Mor structure revealed that it has a classical DNA-binding HTH motif and a dimerization domain. Based on the structure it has been proposed that Mor has to undergo conformational changes to bind DNA. Modelling of C based on the Mor structure revealed that C might also have a dimerization domain and a HTH DNA binding motif. To see if any conformational changes occur in C when it binds DNA, co- crystallization of a C : DNA complex was undertaken. Preliminary structural analysis of the complex revealed that under the crystallization conditions used C protein is bound to its symmetrical binding site using two HTH motifs from two C dimers without inducing any conformational change in itself or the DNA