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.     

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.     

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.     

Solution structure and functional analysis of the cysteine-rich C1 domain of kinase suppressor of Ras (KSR).

Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to promote signal transmission from Raf-1 to MEK and MAPK. All KSR proteins contain a conserved cysteine-rich C1 domain, and studies have implicated this domain in the regulation of KSR1 subcellular localization and function. To further elucidate the biological role of the KSR1 C1 domain, we have determined its three-dimensional solution structure using nuclear magnetic resonance (NMR). We find that while the overall topology of the KSR1 C1 domain is similar to the C1 domains of Raf-1 and PKCgamma, the predicted ligand-binding region and the surface charge distribution are unique. Moreover, by generating chimeric proteins in which these domains have been swapped, we find that the C1 domains of Raf-1, PKCgamma, and KSR1 are not functionally interchangeable. The KSR1 C1 domain does not bind with high affinity or respond biologically to phorbol esters or ceramide, and it does not interact directly with Ras, indicating that the putative ligand(s) for the KSR1 C1 domain are distinct from those that interact with PKCgamma and Raf-1. In addition, our analysis of the chimeric proteins supports the model that Raf-1 is a ceramide-activated kinase and that its C1 domain is involved in the ceramide-mediated response. Finally, our findings demonstrate an absolute requirement of the KSR1 C1 domain in mediating the membrane localization of KSR1, a crucial feature of its scaffolding activity. Together, these results underscore the functional specificity of these important regulatory domains and demonstrate that the structural features of the C1 domains can provide valuable insight into their ligand-binding properties.     

Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin

Ubiquitin is an important cellular signal that targets proteins for degradation or regulates their functions. The previously identified BMSC-UbP protein derived from bone marrow stromal cells contains a ubiquitin-associated (UBA) domain at the C terminus that has been implicated in linking cellular processes and the ubiquitin system. Here, we report the solution NMR structure of the UBA domain of human BMSC-UbP protein and its complex with ubiquitin. The structure determination was facilitated by using a solubility-enhancement tag (SET) GB1, immunoglobulin G binding domain 1 of Streptococcal protein G. The results show that BMSC-UbP UBA domain is primarily comprised of three alpha-helices with a hydrophobic patch defined by residues within the C terminus of helix-1, loop-1, and helix-3. The M-G-I motif is similar to the M/L-G-F/Y motifs conserved in most UBA domains. Chemical shift perturbation study revealed that the UBA domain binds with the conserved five-stranded beta-sheet of ubiquitin via hydrophobic interactions with the dissociation constant (KD) of approximately 17 microM. The structural model of BMSC-UbP UBA domain complexed with ubiquitin was constructed by chemical shift mapping combined with the program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, three residues (Met76, Ile78, and Leu99) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin defined by residues Leu8, Ile44, His68, and Val70. This complex structure may provide clues for BMSC-UbP functions and structural insights into the UBA domains of other ubiquitin-associated proteins that share high sequence homology with BMSC-UbP UBA domain.     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2. STRUCTURED SUMMARY: