Solution structure of the dimeric SAM domain of MAPKKK Ste11 and its interactions with the adaptor protein Ste50 from the budding yeast: implications for Ste11 activation and signal transmission through the Ste50-Ste11 complex

Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae. Ste11/Ste50 binding is mediated by their sterile alpha motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins. Here, we present the solution structure of the SAM domain or residues D37-R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27-Q131. NMR pulse-field-gradient (PFG) and rotational correlation time measurements (tauc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution. The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure. The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5). The dimer structure may also be stabilized by potential salt bridge interactions across the interface. NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain. The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50-Ste11 complex.     

Characterization of the near native conformational states of the SAM domain of Ste11 protein by NMR spectroscopy

The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen‐activated protein kinase cascades. In the current study, urea‐induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions. Proteins 2014; 82:2957–2969. © 2014 Wiley Periodicals, Inc.     

SAM1 domain of SASH1 harbors distinctive structural heterogeneity

The sterile alpha motif (SAM) domains are among the most versatile protein domains in biology, and the variety of the oligomerization states contribute to their diverse roles in many diseases. A better understanding of the structure and dynamics of various SAM domains will provide a scientific basis for drug development targeting them. Here, we used SEC-MALS, HPLC, NMR, and other biophysical techniques to characterize the structural features and dynamics of the SAM1 domain in SASH1. SASH1 is a scaffold protein belonging to the same family as SASH3. Unlike the dimerization seen in SASH3′s SAM domain, our SEC-MALS and SE-HPLC showed that SAM1 exists primarily as a less compact monomer with a minor oligomer. NMR assignment, relaxation, and exchange experiments revealed the presence of both a disordered monomer and a more structured oligomer with multiple timescale exchange regimes in solution. Mutagenesis and SE-HPLC showed that D663A/T664K substitutions in SAM1 increased its oligomerization. In sum, this study is the first to characterize a disordered structure for a SAM domain, provides additional evidence and framework for the diversity of SAM domains, and identifies a region in SAM1 as a potential starting point to further characterize the structural mechanism of oligomerization of the domain.     

Saccharomyces cerevisiae Ste50 binds the MAPKKK Ste11 through a head-to-tail SAM domain interaction

In Saccharomyces cerevisiae, signal transduction through pathways governing mating, osmoregulation, and nitrogen starvation depends upon a direct interaction between the sterile alpha motif (SAM) domains of the Ste11 mitogen-activated protein kinase kinase kinase (MAPKKK) and its regulator Ste50. Previously, we solved the NMR structure of the SAM domain from Ste11 and identified two mutants that diminished binding to the Ste50 SAM domain. Building upon the Ste11 study, we present the NMR structure of the monomeric Ste50 SAM domain and a series of mutants bearing substitutions at surface-exposed hydrophobic amino acid residues. The mid-loop (ML) region of Ste11-SAM, defined by helices H3 and H4 and the end-helix (EH) region of Ste50-SAM, defined by helix H5, were sensitive to substitution, indicating that these two surfaces contribute to the high-affinity interaction. The combination of two mutants, Ste11-SAM-L72R and Ste50-SAM-L69R, formed a high-affinity heterodimer unencumbered by competing homotypic interactions that had prevented earlier NMR studies of the wild-type complex. Yeast bearing mutations that prevented the heterotypic Ste11-Ste50 association in vitro presented signaling defects in the mating and high-osmolarity growth pathways.     

Regulation of clathrin adaptor function in endocytosis: novel role for the SAM domain

During clathrin‐mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin‐binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile α‐motif (SAM) domain and shows a propensity to oligomerize. By co‐immunoprecipitation, Sla1p binds to clathrin and self‐associates in vivo. Mutations in the clathrin‐binding motif that abolish clathrin binding and structure‐based mutations in SHD2 that impede self‐association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin‐mediated endocytosis.     

Solution structure of the Vts1 SAM domain in the presence of RNA

The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the "core" five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition.     

The solution structure of the S.cerevisiae Ste11 MAPKKK SAM domain and its partnership with Ste50

Ste11 is a MAPKKK from Saccharomyces cerevisiae that helps mediate the response to mating pheromone and the ability to thrive in high-salt environments. These diverse functions are facilitated by a direct interaction between the SAM domain of Ste11 with the SAM domain of its regulatory partner, Ste50. We have solved the NMR structure of the Ste11 SAM domain (PDB 1OW5), which reveals a compact, five alpha-helix bundle and a high degree of structural similarity to the Polyhomeotic SAM domain. The combined study of Ste11 SAM rotational correlation times and crosslinking to Ste50-SAM has suggested a mode through which Ste11-SAM oligomerizes and selectively associates with Ste50-SAM. To probe homotypic and heterotypic interations, Ste11-SAM variants each containing a substitution of a surface-exposed hydrophobic residue were constructed. An I59R variant of Ste11-SAM, disrupted binding to Ste50-SAM in vitro. Yeast expressing full-length Ste11-I59R could neither respond to mating pheromone nor thrive in high salt media-demonstrating that the interaction between Ste11 and Ste50 SAM domains is a prerequisite for key signal transduction events.     

Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95

Allosteric regulation is a ubiquitous phenomenon exploited in biological processes to control cells in a myriad of ways. It is also of emerging interest in the design of functional proteins and therapeutics. Even though allostery was proposed over 50 years ago and has been studied intensively from a structural perspective, many key details of allosteric mechanisms remain mysterious. Over the last decade significant attention has been paid to the “dynamic component” of allostery, as opposed to the analysis of rigid structures. Nuclear magnetic resonance spectroscopy and its ability to detect conformationally dynamic processes at atomic resolution have played an important role in expanding our understanding of allosteric mechanisms and opening up new questions. This article focuses on work that highlights how protein dynamics can factor into allosteric processes in distinct ways. Two cases are contrasted. The first considers the “traditionally allosteric” protein CheY, which undergoes a conformational change as a key element of its allostery. The second considers the more rarely observed “dynamic allostery” in a PDZ domain, in which allosteric behavior arises from changes in internal structural dynamics. Interestingly, the dynamic processes in these two contrasting examples occur on different timescales. In the case of the PDZ domain, subsequent experimental and computational work is reviewed to reveal a more complete picture of this interesting case of allostery.     

Structural and functional studies of USP20 ZnF‐UBP domain by NMR

Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia‐inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N‐terminal zinc‐finger ubiquitin binding domain (ZnF‐UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF‐UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF‐UBP domain and investigated its binding properties with mono‐ubiquitin and poly‐ubiquitin (K48‐linked di‐ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF‐UBP domain forms a spherically shaped fold consisting of a central β‐sheet with either one α‐helix or two α‐helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF‐UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF‐UBP's ubiquitin binding capacity was revealed by NMR data‐driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF‐UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di‐glycine motif and the conserved and non‐conserved residues of USP20 ZnF‐UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF‐UBP domain might have a physiological role unrelated to its ubiquitin binding capacity.     

Computer-aided NMR assay for detecting natively folded structural domains

Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space.     

NMR structure of the WIF domain of the human Wnt-inhibitory factor-1

The human Wnt-binding protein Wnt-inhibitory factor-1 (WIF-1) comprises an N-terminal WIF module followed by five EGF-like repeats. Here we report the three-dimensional structure of the WIF domain of WIF-1 determined by NMR spectroscopy. The fold consists of an eight-stranded beta-sandwich reminiscent of the immunoglobulin fold. Residual detergent (Brij-35) used in the refolding protocol was found to bind tightly to the WIF domain. The binding site was identified by intermolecular nuclear Overhauser effects observed between the WIF domain and the alkyl chain of the detergent. The results point to a possible role of WIF domains as a recognition motif of Wnt and Drosophila Hedgehog proteins that are activated by palmitoylation.     

An NMR-based identification of a peptide fragment from the beta-subunit of a G-protein showing specific interactions with the GBB domain of the Ste20 kinase in budding yeast

In mitogen-activated protein kinase (MAPK) cascades of budding yeast, pheromone-induced mating signal is transmitted by interactions between the beta-subunit of a G-protein (G-beta) and the G-beta binding (GBB) domain of Ste20 kinase. Previously, mutational analyses of the beta-subunit of G-protein had identified two critical mutations which abrogate binding of the GBB domain of Ste20. In this work, we have identified, by use of NMR spectroscopy, a peptide fragment from the G-beta that shows specific interactions with the isolated GBB domain of Ste20. A model structure of the Ste20/G-beta complex reveals that the interface of the hetero-complex may be sustained by parallel orientation of two potentially interacting helical segments that are further stabilized by ionic, hydrogen bond, and helix macro-dipole interactions.     

The acidic domain is a unique structural feature of the splicing factor SYNCRIP

The splicing factor SYNCRIP (hnRNP Q) is involved in viral replication, neural morphogenesis, modulation of circadian oscillation, and the regulation of the cytidine deaminase APOBEC1. It consists of three globular RNA‐recognition motifs (RRM) domains flanked by an N‐terminal acid‐rich acidic sequence segment domain (AcD_12–97) and a C‐terminal domain containing an arginine–glycine‐rich sequence motif (RGG/RXG box), which are located near to the N‐ and C‐terminals, respectively. The acid‐rich sequence segment is unique to SYNCRIP and the closely related protein hnRNP R, and is involved in interactions with APOBEC1. Here, we show that while AcD_12–97 does not form a globular domain, structure‐based annotation identified a self‐folding globular domain with an all α‐helix architecture, AcD_24–107. The NMR structure of AcD_24?107 is fundamentally different from previously reported AcD molecular models. In addition to negatively charged surface areas, it contains a large hydrophobic cavity and a positively charged surface area as potential epitopes for intermolecular interactions.     

Binding affinity difference induced by the stereochemistry of the sulfoxide bridge of the cyclic peptide inhibitors of Grb2-SH2 domain: NMR studies for the structural origin

The SAR study on a phage library-derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain indicates that the configuration of the cyclization linkage is crucial for assuming the active binding conformation. When the thioether linkage was oxidized to the two chiral sulfoxides, the R-configured sulfoxide-cyclized peptide displayed 10-30 times more potency than the corresponding S-configured one in binding affinity to the Grb2-SH2 domain. In this paper, the solution structures of such a pair of sulfoxide-bridged cyclic peptide diastereoisomers, i.e., cyclo[CH(2)CO-Gla(1)-L-Y-E-N-V-G-NPG-Y-(R/S)C(O)(10)]-amide, were determined by NMR and molecular dynamics simulation. Results indicate that the consensus sequence of Y(3)-E(4)-N(5)-V(6) in both diastereoisomers adopt a beta-turn conformation; however, the R-configured peptide forms an extended structure with a circular backbone conformation, while the S-configured isomer forms a compact structure with key residues buried inside the molecule. The average root-mean-square deviations were found to be 0.756 and 0.804 A, respectively. It is apparent that the chiral S-->O group played a key role in the solution structures of the sulfoxide-bridged cyclic peptides. The R-sulfoxide group forms an intramolecular hydrogen bond with the C-terminal amide, conferring a more rigid conformation with all residues protruding outside except for Leu2, in which the Gla1 and Tyr3 share an overlapping function as previous SAR studies proposed. Additionally, the extended structure endows a more hydrophilic binding surface of the R-configured peptide to facilitate its capture by its targeted protein. In comparison, the S-configured sulfoxide was embedded inside the ligand peptide leading to a compact structure, in which the essential residues of Gla1, Tyr3, and Asn5 form multiple intramolecular hydrogen bonds resulting in an unfavorable conformational change and a substantial loss of the interaction with the protein. The solution structures disclosed by our NMR and molecular dynamics simulation studies provide a molecular basis for understanding how the chirality of the cyclization linkage remarkably discriminates in terms of the binding affinity, thus advancing the rational design of potent non-phosphorylated inhibitors of Grb2-SH2 domain as antitumor agents.