The Chaperone Activity of the Developmental Small Heat Shock Protein Sip1 Is Regulated by pH-Dependent Conformational Changes.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.

The Chaperone Activity and Substrate Spectrum of Human Small Heat Shock Proteins.

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins. Multicellular organisms contain a large number of different sHsps, raising questions as to whether they function redundantly or are specialized in terms of substrates and mechanism. To gain insight into this issue, we undertook a comparative analysis of the eight major human sHsps on the aggregation of both model proteins and cytosolic lysates under standardized conditions. We discovered that sHsps, which form large oligomers (HspB1/Hsp27, HspB3, HspB4/αA-crystallin, and HspB5/αB-crystallin) are promiscuous chaperones, whereas the chaperone activity of the other sHsps is more substrate-dependent. However, all human sHsps analyzed except HspB7 suppressed the aggregation of cytosolic proteins of HEK293 cells. We identified ∼1100 heat-sensitive HEK293 proteins, 12% of which could be isolated in complexes with sHsps. Analysis of their biochemical properties revealed that most of the sHsp substrates have a molecular mass from 50 to 100 kDa and a slightly acidic pI (5.4-6.8). The potency of the sHsps to suppress aggregation of model substrates is correlated with their ability to form stable substrate complexes; especially HspB1 and HspB5, but also B3, bind tightly to a variety of proteins, whereas fewer substrates were detected in complex with the other sHsps, although these were also efficient in preventing the aggregation of cytosolic proteins.

Hsp90·Cdc37 Complexes with Protein Kinases Form Cooperatively with Multiple Distinct Interaction Sites.

Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37·Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.

BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc.

Nucleotide-Free sB-Raf is Preferentially Bound by Hsp90 and Cdc37 In Vitro.

The molecular chaperone Hsp90 and its cofactor Cdc37 are required for the stability of protein kinases in the cellular environment. Upon pharmacological inhibition of Hsp90, the Hsp90-dependent kinases are degraded quickly by the proteasome. Clear physiological evidence for the formation of heterooligomeric complexes between the chaperone system and its kinase clients exist, but the mechanisms of client processing are still enigmatic. Here, we investigate the interaction of the chaperone system with a stabilized fragment of the Hsp90-dependent protein kinase B-Raf (sB-Raf). sB-Raf is aggregation prone at elevated temperatures. We find that nucleotide binding strongly stabilizes the folded state of sB-Raf and suppresses its aggregation. Also, Cdc37 and Hsp90 in combination can suppress sB-Raf aggregation while forming a ternary complex with the kinase. The presence of nucleotides leads to the dissociation of the kinase from the ternary chaperone complex, implying that the stabilization of the kinase by nucleotides reduces its affinity toward the chaperone machinery. Human Cdc37-Hsp90 complexes can bind to kinase, if the NM domain of the chaperone is present. Nematode Cdc37, which does not require the N-terminal Hsp90 domain for binding, can form a ternary complex with the MC construct of Hsp90, which lacks the aggregation propensity of sB-Raf. Like the full-length complex, this interaction is sensitive to ATP binding to sB-Raf. We thus find that the interaction between sB-Raf and the Hsp90 chaperone system is based on contacts with the M domain of Hsp90, which contributes in forming the ternary complex with CeCdc37 as long as the kinase is not stabilized by nucleotide.

Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion.

To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT(L417M), as proven by SDS-PAGE and size exclusion chromatography. The bifunctional enzyme revealed beneficial kinetics over the two-component system, in particular at low substrate concentration.

Regulatory Implications of Non-Trivial Splicing: Isoform 3 of Rab1A Shows Enhanced Basal Activity and Is Not Controlled by Accessory Proteins.

Alternative splicing often affects structured and highly conserved regions of proteins, generating so called non-trivial splicing variants of unknown structure and cellular function. The human small G-protein Rab1A is involved in the regulation of the vesicle transfer from the ER to Golgi. A conserved non-trivial splice variant lacks nearly 40% of the sequence of the native Rab1A, including most of the regulatory interaction sites. We show that this variant of Rab1A represents a stable and folded protein, which is still able to bind nucleotides and co-localizes with membranes. Nevertheless, it should be mentioned that compared to other wild-typeRabGTPases, the measured nucleotide binding affinities are dramatically reduced in the variant studied. Furthermore, the Rab1A variant forms hetero-dimers with wild-type Rab1A and its presence in the cell enhances the efficiency of alkaline phosphatase secretion. However, this variant shows no specificity for GXP nucleotides, a constantly enhanced GTP hydrolysis activity and is no longer controlled by GEF or GAP proteins, indicating a new regulatory mechanism for the Rab1A cycle via alternative non-trivial splicing.