Structure (v.16, #7)
In This Issue (ix-x).
Sus out Sugars in by Harry John Gilbert (987-989).
In this issue of Structure, show how outer membrane glycan binding proteins contribute to both complex carbohydrate hydrolysis and the presentation of the reaction products to the cognate outer membrane porin.
DegP: a Protein “Death Star” by Damon Huber; Bernd Bukau (989-990).
DegP is both an ATP-independent protease and chaperone in the E. coli periplasm. In a new structural model of DegP recently published in Nature, Krojer et al. suggest that DegP carries out these seemingly opposing roles by assembling into enormous spherical multimers.
Protein-Protein Interactions in the Membrane: Sequence, Structural, and Biological Motifs by David T. Moore; Bryan W. Berger; William F. DeGrado (991-1001).
Single-span transmembrane (TM) helices have structural and functional roles well beyond serving as mere anchors to tether water-soluble domains in the vicinity of the membrane. They frequently direct the assembly of protein complexes and mediate signal transduction in ways analogous to small modular domains in water-soluble proteins. This review highlights different sequence and structural motifs that direct TM assembly and discusses their roles in diverse biological processes. We believe that TM interactions are potential therapeutic targets, as evidenced by natural proteins that modulate other TM interactions and recent developments in the design of TM-targeting peptides.
A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization by Annemarie B. Wöhri; Linda C. Johansson; Pia Wadsten-Hindrichsen; Weixiao Y. Wahlgren; Gerhard Fischer; Rob Horsefield; Gergely Katona; Maria Nyblom; Fredrik Öberg; Gillian Young; Richard J. Cogdell; Niall J. Fraser; Sven Engström; Richard Neutze (1003-1009).
A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.
Ab Initio Folding of Proteins with All-Atom Discrete Molecular Dynamics by Feng Ding; Douglas Tsao; Huifen Nie; Nikolay V. Dokholyan (1010-1018).
Discrete molecular dynamics (DMD) is a rapid sampling method used in protein folding and aggregation studies. Until now, DMD was used to perform simulations of simplified protein models in conjunction with structure-based force fields. Here, we develop an all-atom protein model and a transferable force field featuring packing, solvation, and environment-dependent hydrogen bond interactions. We performed folding simulations of six small proteins (20-60 residues) with distinct native structures by the replica exchange method. In all cases, native or near-native states were reached in simulations. For three small proteins, multiple folding transitions are observed, and the computationally characterized thermodynamics are in qualitative agreement with experiments. The predictive power of all-atom DMD highlights the importance of environment-dependent hydrogen bond interactions in modeling protein folding. The developed approach can be used for accurate and rapid sampling of conformational spaces of proteins and protein-protein complexes and applied to protein engineering and design of protein-protein interactions.
Structural Basis for Dimerization in DNA Recognition by Gal4 by Manqing Hong; Mary X. Fitzgerald; Sandy Harper; Cheng Luo; David W. Speicher; Ronen Marmorstein (1019-1026).
Gal4 is a Zn2Cys6 binuclear cluster containing transcription factor that binds DNA as a homodimer and can activate transcription by interacting with the mutant Gal11P protein. Although structures have been reported of the Gal4 dimerization domain and the binuclear cluster domain bound to DNA as a dimer, the structure of the “complete” Gal4 dimer bound to DNA has not previously been described. Here we report the structure of a complete Gal4 dimer bound to DNA and additional biochemical studies to address the molecular basis for Gal4 dimerization in DNA binding. We find that Gal4 dimerization on DNA is mediated by an intertwined helical bundle that deviates significantly from the solution NMR structure of the free dimerization domain. Associated biochemical studies show that the dimerization domain of Gal4 is important for DNA binding and protein thermostability. We also map the interaction surface of the Gal4 dimerization domain with Gal11P.
Crystal Structure of the Outer Membrane Protein OpdK from Pseudomonas aeruginosa by Shyamasri Biswas; Mohammad M. Mohammad; Liviu Movileanu; Bert van den Berg (1027-1035).
In Gram-negative bacteria that do not have porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. We report here the X-ray crystal structure of OpdK, an OprD family member implicated in the uptake of vanillate and related small aromatic acids. The OpdK structure reveals a monomeric, 18-stranded β barrel with a kidney-shaped central pore. The OpdK pore constriction is relatively wide for a substrate-specific channel (∼8 Å diameter), and it is lined by a positively charged patch of arginine residues on one side and an electronegative pocket on the opposite side—features likely to be important for substrate selection. Single-channel electrical recordings of OpdK show binding of vanillate to the channel, and they suggest that OpdK forms labile trimers in the outer membrane. Comparison of the OpdK structure with that of Pseudomonas aeruginosa OprD provides the first qualitative insights into the different substrate specificities of these closely related channels.
Keywords: PROTEINS; MICROBIO;
Analysis of the Staphylococcus aureus DgkB Structure Reveals a Common Catalytic Mechanism for the Soluble Diacylglycerol Kinases by Darcie J. Miller; Agoston Jerga; Charles O. Rock; Stephen W. White (1036-1046).
Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg2+ site and an associated Asp•water•Mg2+ network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp•water•Mg2+ network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.
Keywords: PROTEINS; SIGNALING;
Beyond Induced-Fit Receptor-Ligand Interactions: Structural Changes that Can Significantly Extend Bond Lifetimes by Lina M. Nilsson; Wendy E. Thomas; Evgeni V. Sokurenko; Viola Vogel (1047-1058).
While the lifetime of conventional receptor-ligand interactions is shortened by tensile mechanical force, some recently discovered interactions, termed catch bonds, can be strengthened by force. Motivated by the search for the underpinning structural mechanisms, we here explore the structural dynamics of the binding site of the bacterial adhesive protein FimH by molecular dynamics and steered molecular dynamics. While the crystal structure of only one FimH conformation has been reported so far, we describe two distinctively different conformations of the mannose-bound FimH binding site. Force-induced dissociation was slowed when the mannose ring rotated such that additional force-bearing hydrogen bonds formed with the base of the FimH binding pocket. The lifetime of the complex was further enhanced significantly by rigidifying this base. We finally show how even sub-angstrom spatial alterations of the hydrogen bonding pattern within the base can lead to significantly decreased bond lifetimes.
Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase by Hasan Demirci; Steven T. Gregory; Albert E. Dahlberg; Gerwald Jogl (1059-1066).
Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal α-amino group and the ɛ-amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal α-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.
Keywords: RNA; PROTEINS;
The Interplay of Functional Tuning, Drug Resistance, and Thermodynamic Stability in the Evolution of the M2 Proton Channel from the Influenza A Virus by Amanda L. Stouffer; Chunlong Ma; Lidia Cristian; Yuki Ohigashi; Robert A. Lamb; James D. Lear; Lawrence H. Pinto; William F. DeGrado (1067-1076).
We explore the interplay between amino acid sequence, thermodynamic stability, and functional fitness in the M2 proton channel of influenza A virus. Electrophysiological measurements show that drug-resistant mutations have minimal effects on M2’s specific activity, and suggest that resistance is achieved by altering a binding site within the pore rather than a less direct allosteric mechanism. In parallel, we measure the effects of these mutations on the free energy of assembling the homotetrameric transmembrane pore from monomeric helices in micelles and bilayers. Although there is no simple correlation between the evolutionary fitness of the mutants and their stability, all variants formed more stable tetramers in bilayers, and the least-fit mutants showed the smallest increase in stability upon moving from a micelle to a bilayer environment. We speculate that the folding landscape of a micelle is rougher than that of a bilayer, and more accommodating of conformational variations in nonoptimized mutants.
Keywords: PROTEINS; MICROBIO;
Structural Basis for the Recognition of Histone H4 by the Histone-Chaperone RbAp46 by Natalia V. Murzina; Xue-Yuan Pei; Wei Zhang; Mike Sparkes; Jose Vicente-Garcia; J. Venkatesh Pratap; Stephen H. McLaughlin; Tom Rolef Ben-Shahar; Alain Verreault; Ben F. Luisi; Ernest D. Laue (1077-1085).
RbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed β propeller structure and binds histone H4 in a groove formed between an N-terminal α helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.
Keywords: PROTEINS; DNA;
RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer Gβ1γ2 by Christopher A. Johnston; Adam J. Kimple; Patrick M. Giguère; David P. Siderovski (1086-1094).
This article has been retracted at the request of the Authors.In this paper, a co-crystal structure was described of the G-protein heterodimer Gβ1γ2 bound to a C-terminal peptide from the parathyroid hormone receptor-1 (PTH1R) at 3.0 Å resolution (PDB id 2QNS). A subsequent refinement was later deposited in the Protein Data Bank (PDB id 3KJ5). While this structure represents a new crystal form of the Gβ1γ2 heterodimer, because of the lack of clear and continuous electron density for the receptor peptide in the complex structure, the paper is being retracted. We apologize for any confusion this may have caused.
The tRNA-Induced Conformational Activation of Human Mitochondrial Phenylalanyl-tRNA Synthetase by Liron Klipcan; Inna Levin; Naama Kessler; Nina Moor; Igal Finarov; Mark Safro (1095-1104).
All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 Å resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the α and anticodon binding domain (ABD) of the bacterial β subunit of (αβ)2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNAPhe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNAPhe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through ∼160°) of the ABD upon tRNA binding.
Starch Catabolism by a Prominent Human Gut Symbiont Is Directed by the Recognition of Amylose Helices by Nicole M. Koropatkin; Eric C. Martens; Jeffrey I. Gordon; Thomas J. Smith (1105-1115).
The human gut microbiota performs functions that are not encoded in our Homo sapiens genome, including the processing of otherwise undigestible dietary polysaccharides. Defining the structures of proteins involved in the import and degradation of specific glycans by saccharolytic bacteria complements genomic analysis of the nutrient-processing capabilities of gut communities. Here, we describe the atomic structure of one such protein, SusD, required for starch binding and utilization by Bacteroides thetaiotaomicron, a prominent adaptive forager of glycans in the distal human gut microbiota. The binding pocket of this unique α-helical protein contains an arc of aromatic residues that complements the natural helical structure of starch and imposes this conformation on bound maltoheptaose. Furthermore, SusD binds cyclic oligosaccharides with higher affinity than linear forms. The structures of several SusD/oligosaccharide complexes reveal an inherent ligand recognition plasticity dominated by the three-dimensional conformation of the oligosaccharides rather than specific interactions with the composite sugars.
Keywords: PROTEINS; MICROBIO;
The Crystal Structure of the Ran-Nup153ZnF2 Complex: a General Ran Docking Site at the Nuclear Pore Complex by Nils Schrader; Carolin Koerner; Katja Koessmeier; Jan-Amadé Bangert; Alfred Wittinghofer; Raphael Stoll; Ingrid R. Vetter (1116-1125).
Nucleoporin (Nup) 153 is a highly mobile, multifunctional, and essential nuclear pore protein. It contains four zinc finger motifs that are thought to be crucial for the regulation of transport-receptor/cargo interactions via their binding to the small guanine nucleotide binding protein, Ran. We found this interaction to be independent of the phoshorylation state of the nucleotide. Ran binds with the highest affinity to the second zinc finger motif of Nup153 (Nup153ZnF2). Here we present the crystal structure of this complex, revealing a new type of Ran-Ran interaction partner interface together with the solution structure of Nup153ZnF2. According to our complex structure, Nup153ZnF2 binding to Ran excludes the formation of a Ran-importin-β complex. This finding suggests a local Nup153-mediated Ran reservoir at the nucleoplasmic distal ring of the nuclear pore, where nucleotide exchange may take place in a ternary Nup153-Ran-RCC1 complex, so that import complexes are efficiently terminated.
Single Copies of Sec61 and TRAP Associate with a Nontranslating Mammalian Ribosome by Jean-François Ménétret; Ramanujan S. Hegde; Mike Aguiar; Steven P. Gygi; Eunyong Park; Tom A. Rapoport; Christopher W. Akey (1126-1137).
During cotranslational protein translocation, the ribosome associates with a membrane channel, formed by the Sec61 complex, and recruits the translocon-associated protein complex (TRAP). Here we report the structure of a ribosome-channel complex from mammalian endoplasmic reticulum in which the channel has been visualized at 11 Å resolution. In this complex, single copies of Sec61 and TRAP associate with a nontranslating ribosome and this stoichiometry was verified by quantitative mass spectrometry. A bilayer-like density surrounds the channel and can be attributed to lipid and detergent. The crystal structure of an archaeal homolog of the Sec61 complex was then docked into the map. In this model, two cytoplasmic loops of Sec61 may interact with RNA helices H6, H7, and H50, while the central pore is located below the ribosome tunnel exit. Hence, this copy of Sec61 is positioned to capture and translocate the nascent chain. Finally, we show that mammalian and bacterial ribosome-channel complexes have similar architectures.
Keywords: PROTEINS; DNA;
The SNARE Complex from Yeast Is Partially Unstructured on the Membrane by Zengliu Su; Yuji Ishitsuka; Taekjip Ha; Yeon-Kyun Shin (1138-1146).
Molecular recognition between cognate SNAREs leads to the formation of a four-helix bundle, which facilitates vesicle docking and membrane fusion. For a SNARE system involved in trafficking in yeast, target membrane (t-) SNARE Sso1p and vesicle associated (v-) SNARE Snc2p contribute one SNARE motif each, whereas another t-SNARE (Sec9) donates two N-terminal and C-terminal SNARE motifs (SN1 and SN2) to the helical bundle. By use of EPR, it is found that SN2 has a tendency to be uncoiled, leaving a significant population of the SNARE complexes to be partially unstructured on the membrane. In sharp contrast, SN2 is fully engaged in the four-helix bundle when removed from the membrane, showing that the membrane is the main destabilizing factor. Helix-breaking proline mutations in SN2 did not affect the rate of docking but reduced the rate of lipid mixing significantly, indicating that SN2 plays an essential role in activating the transition from docking to fusion.
Force Generation in Kinesin Hinges on Cover-Neck Bundle Formation by Wonmuk Hwang; Matthew J. Lang; Martin Karplus (1147).