Santiago Martínez-Lumbreras

Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Background The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Methodology and Principal Findings SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. Significance This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.

Structural and Functional Insights into Small, Glutamine-Rich, Tetratricopeptide Repeat Protein Alpha

The small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA) is an emerging player in the quality control of secretory and membrane proteins mislocalized to the cytosol, with established roles in tail-anchored (TA) membrane protein biogenesis. SGTA consists of three structural domains with individual functions, an N-terminal dimerization domain that assists protein sorting pathways, a central tetratricopeptide repeat (TPR) domain that mediates interactions with heat-shock proteins, proteasomal, and hormonal receptors, and viral proteins, and a C-terminal glutamine rich region that binds hydrophobic substrates. SGTA has been linked to viral lifecycles and hormone receptor signaling, with implications in the pathogenesis of various disease states. Thus far, a range of biophysical techniques have been employed to characterize SGTA structure in some detail, and to investigate its interactions with binding partners in different biological contexts. A complete description of SGTA structure, together with further investigation into its function as a co-chaperone involved quality control, could provide us with useful insights into its role in maintaining cellular proteostasis, and broaden our understanding of mechanisms underlying associated pathologies. This review describes how some structural features of SGTA have been elucidated, and what this has uncovered about its cellular functions. A brief background on the structure and function of SGTA is given, highlighting its importance to biomedicine and related fields. The current level of knowledge and what remains to be understood about the structure and function of SGTA is summarized, discussing the potential direction of future research.

Structural and functional insights into the E3 ligase, RNF126.

RNF126 is an E3 ubiquitin ligase that collaborates with the BAG6 sortase complex to ubiquitinate hydrophobic substrates in the cytoplasm that are destined for proteasomal recycling. Composed of a trimeric complex of BAG6, TRC35 and UBL4A the BAG6 sortase is also associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Here we solve the solution structure of the RNF126 zinc finger domain in complex with the BAG6 UBL domain. We also characterise an interaction between RNF126 and UBL4A and analyse the competition between SGTA and RNF126 for the N-terminal BAG6 binding site. This work sheds light on the sorting mechanism of the BAG6 complex and its accessory proteins which, together, decide the fate of stray hydrophobic proteins in the aqueous cytoplasm.

SGTA interacts with the proteasomal ubiquitin receptor Rpn13 via a carboxylate clamp mechanism

The fate of secretory and membrane proteins that mislocalize to the cytosol is decided by a collaboration between cochaperone SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the BAG6 complex, whose operation relies on multiple transient and subtly discriminated interactions with diverse binding partners. These include chaperones, membrane-targeting proteins and ubiquitination enzymes. Recently a direct interaction was discovered between SGTA and the proteasome, mediated by the intrinsic proteasomal ubiquitin receptor Rpn13. Here, we structurally and biophysically characterize this binding and identify a region of the Rpn13 C-terminal domain that is necessary and sufficient to facilitate it. We show that the contact occurs through a carboxylate clamp-mediated molecular recognition event with the TPR domain of SGTA, and provide evidence that the interaction can mediate the association of Rpn13 and SGTA in a cellular context.

Structure and Interactions of the TPR Domain of Sgt2 with Yeast Chaperones and Ybr137wp

Small glutamine-rich tetratricopeptide repeat-containing protein 2 (Sgt2) is a multi-module co-chaperone involved in several protein quality control pathways. The TPR domain of Sgt2 and several other proteins, including SGTA, Hop, and CHIP, is a highly conserved motif known to form transient complexes with molecular chaperones such as Hsp70 and Hsp90. In this work, we present the first high resolution crystal structures of Sgt2_TPR alone and in complex with a C-terminal peptide PTVEEVD from heat shock protein, Ssa1. Using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we demonstrate that Sgt2_TPR interacts with peptides corresponding to the C-termini of Ssa1, Hsc82, and Ybr137wp with similar binding modes and affinities.

A Novel RNA Polymerase-binding Protein that interacts with a Sigma-Factor Docking Site

Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σF to create a negative feedback loop that inhibits σF‐directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σF‐directed gene transcription. Based on pull‐down experiments, chemical crosslinking, bacterial two‐hybrid experiments and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled‐coil region of the β′ subunit. The coiled‐coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σF to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti‐σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σF by competing for binding to the β′ coiled‐coil.

Structural and Functional Insights into Bacillus subtilis Sigma Factor Inhibitor, CsfB

Summary Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition

Abstract Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/β domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.