Structure (v.14, #4)
I Siah Substrate! by Douglas J. Briant; Derek F. Ceccarelli; Frank Sicheri (627-628).
Proteins are targeted to the E3 RING ubiquitin ligase Siah through a PxAxVxP degron motif. In this issue of Structure, present the structural basis by which Siah recognizes its degron with high affinity and specificity.
A Monotopic Membrane Protein Goes Solo by Andrea Mattevi (628-629).
Carnitine palmitoyltransferases (CPTs) are part of the enzymatic system that imports fatty acids into mitochondria. The crystal structure of rat CPT-2 by (this issue of Structure) reveals a Y-shaped tunnel for binding the CoA and acyl-carnitine substrates and a hydrophobic insert mediating membrane association.
Shedding UV Light on the Phase Problem by Joel L. Sussman; Israel Silman (629-630).
In this issue of Structure, describe the use of UV-induced radiation damage (UV-RIP) to solve the phase problem for proteins, employing single-wavelength X-ray radiation, without the need for derivatization. This should also permit data collection for many proteins on home sources, without travel to a synchrotron.
ESCRT Complexes Assembled and GLUEd by Sudharshan Eathiraj; David G. Lambright (631-632).
The ESCRT-I, -II, and -III complexes act sequentially to sort monoubiquitinated transmembrane proteins into multivesicular bodies for targeted degradation in the lysosome. Two papers published in a recent issue of Cell provide insights into the structural organization and functional interactions of the ESCRT-I complex and ESCRT-II GLUE domain.
Bridging Conformational Dynamics and Function Using Single-Molecule Spectroscopy by Sua Myong; Benjamin C. Stevens; Taekjip Ha (633-643).
In a typical structure-function relation study, the primary structure of proteins or nucleic acids is changed by mutagenesis and its functional effect is measured via biochemical means. Single-molecule spectroscopy has begun to give a whole new meaning to the “structure-function relation” by measuring the real-time conformational changes of individual biological macromolecules while they are functioning. This review discusses a few recent examples: untangling internal chemistry and conformational dynamics of a ribozyme, branch migration landscape of a Holliday junction at a single-step resolution, tRNA selection and dynamics in a ribosome, repetitive shuttling and snapback of a helicase, and discrete rotation of an ATP synthase.
HIV-1 Coreceptor Selectivity: Structural Analogy between HIV-1 V3 Regions and Chemokine β-Hairpins Is Not the Explanation by Holger Scheib; Peter Sperisen; Oliver Hartley (645-647).
Response to Matters Arising by Osnat Rosen; Abraham O. Samson; Michal Sharon; Susan Zolla-Pazner; Jacob Anglister (649-651).
Protease Accessibility Laddering: A Proteomic Tool for Probing Protein Structure by Svetlana Dokudovskaya; Rosemary Williams; Damien Devos; Andrej Sali; Brian T. Chait; Michael P. Rout (653-660).
Limited proteolysis is widely used in biochemical and crystallographic studies to determine domain organization, folding properties, and ligand binding activities of proteins. The method has limitations, however, due to the difficulties in obtaining sufficient amounts of correctly folded proteins and in interpreting the results of the proteolysis. A new limited proteolysis method, named protease accessibility laddering (PAL), avoids these complications. In PAL, tagged proteins are purified on magnetic beads in their natively folded state. While attached to the beads, proteins are probed with proteases. Proteolytic fragments are eluted and detected by immunoblotting with antibodies against the tag (e.g., Protein A, GFP, and 6×His). PAL readily detects domain boundaries and flexible loops within proteins. A combination of PAL and comparative protein structure modeling allows characterization of previously unknown structures (e.g., Sec31, a component of the COPII coated vesicle). PAL's high throughput should greatly facilitate structural genomic and proteomic studies.
The Endosome-Associated Protein Hrs Is Hexameric and Controls Cargo Sorting as a “Master Molecule” by Lee Pullan; Srinivas Mullapudi; Zhong Huang; Philip R. Baldwin; Christopher Chin; Wei Sun; Susan Tsujimoto; Steven J. Kolodziej; James K. Stoops; J. Ching Lee; M. Neal Waxham; Andrew J. Bean; Pawel A. Penczek (661-671).
The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a “master molecule” that presents multiple sites for protein binding.
Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins by Toshimitsu Kawate; Eric Gouaux (673-681).
Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.
Flexibility and Conformational Entropy in Protein-Protein Binding by Raik Grünberg; Michael Nilges; Johan Leckner (683-693).
To better understand the interplay between protein-protein binding and protein dynamics, we analyzed molecular dynamics simulations of 17 protein-protein complexes and their unbound components. Complex formation does not restrict the conformational freedom of the partner proteins as a whole, but, rather, it leads to a redistribution of dynamics. We calculate the change in conformational entropy for seven complexes with quasiharmonic analysis. We see significant loss, but also increased or unchanged conformational entropy. Where comparison is possible, the results are consistent with experimental data. However, stringent error estimates based on multiple independent simulations reveal large uncertainties that are usually overlooked. We observe substantial gains of pseudo entropy in individual partner proteins, and we observe that all complexes retain residual stabilizing intermolecular motions. Consequently, protein flexibility has an important influence on the thermodynamics of binding and may disfavor as well as favor association. These results support a recently proposed unified model for flexible protein-protein association.
Elucidation of the Substrate Binding Site of Siah Ubiquitin Ligase by Colin M. House; Nancy C. Hancock; Andreas Möller; Brett A. Cromer; Victor Fedorov; David D.L. Bowtell; Michael W. Parker; Galina Polekhina (695-701).
The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two β strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target.
Crystal Structure of the HP1-EMSY Complex Reveals an Unusual Mode of HP1 Binding by Ying Huang; Michael P. Myers; Rui-Ming Xu (703-712).
Heterochromatin protein-1 (HP1) plays an essential role in both the assembly of higher-order chromatin structure and epigenetic inheritance. The C-terminal chromo shadow domain (CSD) of HP1 is responsible for homodimerization and interaction with a number of chromatin-associated nonhistone proteins, including EMSY, which is a BRCA2-interacting protein that has been implicated in the development of breast and ovarian cancer. We have determined the crystal structure of the HP1β CSD in complex with the N-terminal domain of EMSY at 1.8 Å resolution. Surprisingly, the structure reveals that EMSY is bound by two HP1 CSD homodimers, and the binding sequences differ from the consensus HP1 binding motif PXVXL. This structural information expands our understanding of HP1 binding specificity and provides insights into interactions between HP1 homodimers that are likely to be important for heterochromatin formation.
Keywords: DNA; CELLCYCLE;
The Crystal Structure of Carnitine Palmitoyltransferase 2 and Implications for Diabetes Treatment by Arne C. Rufer; Ralf Thoma; Jörg Benz; Martine Stihle; Bernard Gsell; Elodie De Roo; David W. Banner; Francis Mueller; Odile Chomienne; Michael Hennig (713-723).
Carnitine palmitoyltransferases 1 and 2 (CPTs) facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. We report here the 1.6 Å resolution structure of the full-length mitochondrial membrane protein CPT-2. The structure of CPT-2 in complex with the generic CPT inhibitor ST1326 ([R]-N-[tetradecylcarbamoyl]-aminocarnitine), a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for diabetes mellitus treatment, was solved at 2.5 Å resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of antidiabetic drugs. We identify a sequence insertion found in CPT-2 that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2.
Keywords: HUMDISEASE; CHEMBIO;
Structure of a Leu3-DNA Complex: Recognition of Everted CGG Half-Sites by a Zn2Cys6 Binuclear Cluster Protein by Mary X. Fitzgerald; Jeannie R. Rojas; John M. Kim; Gunter B. Kohlhaw; Ronen Marmorstein (725-735).
Gal4 is the prototypical Zn2Cys6 binuclear cluster transcriptional regulator that binds as a homodimer to DNA containing inverted CGG half-sites. Leu3, a member of this protein family, binds to everted (opposite polarity to inverted) CGG half-sites, and an H50C mutation within the Leu3 Zn2Cys6 binuclear motif abolishes its transcriptional repression function without impairing DNA binding. We report the X-ray crystal structures of DNA complexes with Leu3 and Leu3(H50C) and solution DNA binding studies of selected Leu3 mutant proteins. These studies reveal the molecular details of everted CGG half-site recognition, and suggest a role for the H50C mutation in transcriptional repression. Comparison with the Gal4-DNA complex shows an unexpected conservation in the DNA recognition mode of inverted and everted CGG half-sites, and points to a critical function of a linker region between the Zn2Cys6 binuclear cluster and dimerization regions in DNA binding specificity. Broader implications of these findings are discussed.
The Structure of a Ketoreductase Determines the Organization of the β-Carbon Processing Enzymes of Modular Polyketide Synthases by Adrian T. Keatinge-Clay; Robert M. Stroud (737-748).
The structure of the ketoreductase (KR) from the first module of the erythromycin synthase with NADPH bound was solved to 1.79 Å resolution. The 51 kDa domain has two subdomains, each similar to a short-chain dehydrogenase/reductase (SDR) monomer. One subdomain has a truncated Rossmann fold and serves a purely structural role stabilizing the other subdomain, which catalyzes the reduction of the β-carbonyl of a polyketide and possibly the epimerization of an α-substituent. The structure enabled us to define the domain boundaries of KR, the dehydratase (DH), and the enoylreductase (ER). It also constrains the three-dimensional organization of these domains within a module, revealing that KR does not make dimeric contacts across the 2-fold axis of the module. The quaternary structure elucidates how substrates are shuttled between the active sites of polyketide synthases (PKSs), as well as related fatty acid synthases (FASs), and suggests how domains can be swapped to make hybrid synthases that produce novel polyketides.
Keywords: MICROBIO; CHEMBIO;
An Incoming Nucleotide Imposes an anti to syn Conformational Change on the Templating Purine in the Human DNA Polymerase-ι Active Site by Deepak T. Nair; Robert E. Johnson; Louise Prakash; Satya Prakash; Aneel K. Aggarwal (749-755).
Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPolι binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP “pushes” templates A and G from the anti to the syn conformation dictated by a rigid hPolι active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.
Magnesium-Induced Assembly of a Complete DNA Polymerase Catalytic Complex by Vinod K. Batra; William A. Beard; David D. Shock; Joseph M. Krahn; Lars C. Pedersen; Samuel H. Wilson (757-766).
The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3′-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3′-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase β lacking the 3′-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3′ on the αP of the incoming nucleotide.
Biochemical Characterization and Crystal Structure of Synechocystis Arogenate Dehydrogenase Provide Insights into Catalytic Reaction by Pierre Legrand; Renaud Dumas; Marlene Seux; Pascal Rippert; Raimond Ravelli; Jean-Luc Ferrer; Michel Matringe (767-776).
The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 Å. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique α-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction.
Structural Basis for NHERF Recognition by ERM Proteins by Shin-ichi Terawaki; Ryoko Maesaki; Toshio Hakoshima (777-789).
The Na+/H+ exchanger regulatory factor (NHERF) is a key adaptor protein involved in the anchoring of ion channels and receptors to the actin cytoskeleton through binding to ERM (ezrin/radixin/moesin) proteins. NHERF binds the FERM domain of ERM proteins, although NHERF has no signature Motif-1 sequence for FERM binding found in adhesion molecules. The crystal structures of the radixin FERM domain complexed with the NHERF-1 and NHERF-2 C-terminal peptides revealed a peptide binding site of the FERM domain specific for the 13 residue motif MDWxxxxx(L/I)Fxx(L/F) (Motif-2), which is distinct from Motif-1. This Motif-2 forms an amphipathic α helix for hydrophobic docking to subdomain C of the FERM domain. This docking causes induced-fit conformational changes in subdomain C and affects binding to adhesion molecule peptides, while the two binding sites are not overlapped. Our studies provide structural paradigms for versatile ERM linkages between membrane proteins and the cytoskeleton.
Phasing Macromolecular Structures with UV-Induced Structural Changes by Max H. Nanao; Raimond B.G. Ravelli (791-800).
Experimental phasing of macromolecular crystal structures relies on the accurate measurement of two or more sets of reflections from isomorphous crystals, where the scattering power of a few atoms is different for each set. Recently, it was demonstrated that X-ray-induced intensity differences can also contain phasing information, exploiting specific structural changes characteristic of X-ray damage. This method (radiation damage-induced phasing; RIP) has the advantage that it can be performed on a single crystal of the native macromolecule. However, a drawback is that X-rays introduce many small changes to both solvent and macromolecule. In this study, ultraviolet (UV) radiation has been used to induce specific changes in the macromolecule alone, leading to a larger contrast between radiation-susceptible and nonsusceptible sites. Unlike X-ray RIP, UV RIP does not require the use of a synchrotron. The method has been demonstrated for a series of macromolecules.