Structure (v.16, #4)
In This Issue (ix-x).
NotI Is Not Boring by Giedre Tamulaitiene; Virginijus Siksnys (497-498).
Crystal structures of the restriction endonuclease NotI in free and DNA bound forms, presented in this issue of Structure (), provide a unique insight into the structural details of 8 base pair sequence recognition by the restriction enzyme.
Exit Biology: Battle for the Nascent Chain by Maria Selmer; Anders Liljas (498-500).
Ban and coworkers at ETH are step-by-step widening the perspective of the actions on the ribosome (). The last one in the series is the localization of the binding site of peptide deformylase at the mouth of the polypeptide exit tunnel.
Characterization of a Trifunctional Mimivirus mRNA Capping Enzyme and Crystal Structure of the RNA Triphosphatase Domain by Delphine Benarroch; Paul Smith; Stewart Shuman (501-512).
The RNA triphosphatase (RTPase) components of the mRNA capping apparatus are a bellwether of eukaryal taxonomy. Fungal and protozoal RTPases belong to the triphosphate tunnel metalloenzyme (TTM) family, exemplified by yeast Cet1. Several large DNA viruses encode metal-dependent RTPases unrelated to the cysteinyl-phosphatase RTPases of their metazoan host organisms. The origins of DNA virus RTPases are unclear because they are structurally uncharacterized. Mimivirus, a giant virus of amoeba, resembles poxviruses in having a trifunctional capping enzyme composed of a metal-dependent RTPase module fused to guanylyltransferase (GTase) and guanine-N7 methyltransferase domains. The crystal structure of mimivirus RTPase reveals a minimized tunnel fold and an active site strikingly similar to that of Cet1. Unlike homodimeric fungal RTPases, mimivirus RTPase is a monomer. The mimivirus TTM-type RTPase-GTase fusion resembles the capping enzymes of amoebae, providing evidence that the ancestral large DNA virus acquired its capping enzyme from a unicellular host.
Structure Prediction of Domain Insertion Proteins from Structures of Individual Domains by Monica Berrondo; Marc Ostermeier; Jeffrey J. Gray (513-527).
Multidomain proteins continue to be a major challenge in protein structure prediction. Here we present a Monte Carlo (MC) algorithm, implemented within Rosetta, to predict the structure of proteins in which one domain is inserted into another. Three MC moves combine rigid-body and loop movements to search the constrained conformation by structure disruption and subsequent repair of chain breaks. Local searches find that the algorithm samples and recovers near-native structures consistently. Further global searches produced top-ranked structures within 5 Å in 31 of 50 cases in low-resolution mode, and refinement of top-ranked low-resolution structures produced models within 2 Å in 21 of 50 cases. Rigid-body orientations were often correctly recovered despite errors in linker conformation. The algorithm is broadly applicable to de novo structure prediction of both naturally occurring and engineered domain insertion proteins.
Multiple States of a Nucleotide-Bound Group 2 Chaperonin by Daniel K. Clare; Scott Stagg; Joel Quispe; George W. Farr; Arthur L. Horwich; Helen R. Saibil (528-534).
Chaperonin action is controlled by cycles of nucleotide binding and hydrolysis. Here, we examine the effects of nucleotide binding on an archaeal group 2 chaperonin. In contrast to the ordered apo state of the group 1 chaperonin GroEL, the unliganded form of the homo-16-mer Methanococcus maripaludis group 2 chaperonin is very open and flexible, with intersubunit contacts only in the central double belt of equatorial domains. The intermediate and apical domains are free of contacts and deviate significantly from the overall 8-fold symmetry. Nucleotide binding results in three distinct, ordered 8-fold symmetric conformations—open, partially closed, and fully closed. The partially closed ring encloses a 40% larger volume than does the GroEL-GroES folding chamber, enabling it to encapsulate proteins up to 80 kDa, in contrast to the fully closed form, whose cavities are 20% smaller than those of the GroEL-GroES chamber.
Structure of the Mammalian 80S Ribosome at 8.7 Å Resolution by Preethi Chandramouli; Maya Topf; Jean-François Ménétret; Narayanan Eswar; Jamie J. Cannone; Robin R. Gutell; Andrej Sali; Christopher W. Akey (535-548).
In this paper, we present a structure of the mammalian ribosome determined at ∼8.7 Å resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for ∼20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.
Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity by Yogesh K. Gupta; Deepak T. Nair; Robin P. Wharton; Aneel K. Aggarwal (549-557).
Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycBreverse and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, in effect, acting as a “spacer.” The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.
Structures of the Rare-Cutting Restriction Endonuclease NotI Reveal a Unique Metal Binding Fold Involved in DNA Binding by Abigail R. Lambert; Django Sussman; Betty Shen; Robert Maunus; Jay Nix; James Samuelson; Shuang-Yong Xu; Barry L. Stoddard (558-569).
The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5′-GCGGCCGC-3′, has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.
The Evolutionarily Conserved Family of Cyanovirin-N Homologs: Structures and Carbohydrate Specificity by Leonardus M.I. Koharudin; Arturo R. Viscomi; Jun-Goo Jee; Simone Ottonello; Angela M. Gronenborn (570-584).
Solution structures for three members of the recently discovered cyanovirin-N (CV-N) homolog family of lectins have been determined. Cyanovirin-N homologs (CVNHs) from Tuber borchii, Ceratopteris richardii, and Neurospora crassa, representing each of the three phylogenetic groups, were selected. All proteins exhibit the same fold, and the overall structures resemble that of the founding member of the family, CV-N, albeit with noteworthy differences in loop conformation and detailed local structure. Since no data are available regarding the proteins' function or their natural ligands, extensive carbohydrate-binding studies were conducted. We delineated ligand-binding sites on all three proteins by nuclear magnetic resonance and identified which sugars interact by array screening. The number and location of binding sites vary for the three proteins, and different ligand specificities exist. Potential physiological roles for two family members, TbCVNH and NcCVNH, were probed in nutrition deprivation experiments that suggest a possible involvement of these proteins in lifestyle-related responses.
The GTPase Cycle of the Chloroplast Import Receptors Toc33/Toc34: Implications from Monomeric and Dimeric Structures by Patrick Koenig; Mislav Oreb; Anja Höfle; Sabine Kaltofen; Karsten Rippe; Irmgard Sinning; Enrico Schleiff; Ivo Tews (585-596).
Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is—in the dimer—positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.
Keywords: PROTEINS; CELLBIO;
Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa-Factor VIIIa Complex by Jacky Chi Ki Ngo; Mingdong Huang; David A. Roth; Barbara C. Furie; Bruce Furie (597-606).
Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca2+ and two Cu2+ ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.
Crystal Structure of the CaV2 IQ Domain in Complex with Ca2+/Calmodulin: High-Resolution Mechanistic Implications for Channel Regulation by Ca2+ by Masayuki X. Mori; Craig W. Vander Kooi; Daniel J. Leahy; David T. Yue (607-620).
Calmodulin (CaM) regulation of Ca2+ channels is central to Ca2+ signaling. CaV1 versus CaV2 classes of these channels exhibit divergent forms of regulation, potentially relating to customized CaM/IQ interactions among different channels. Here we report the crystal structures for the Ca2+/CaM IQ domains of both CaV2.1 and CaV2.3 channels. These highly similar structures emphasize that major CaM contacts with the IQ domain extend well upstream of traditional consensus residues. Surprisingly, upstream mutations strongly diminished CaV2.1 regulation, whereas downstream perturbations had limited effects. Furthermore, our CaV2 structures closely resemble published Ca2+/CaM-CaV1.2 IQ structures, arguing against CaV1/2 regulatory differences based solely on contrasting CaM/IQ conformations. Instead, alanine scanning of the CaV2.1 IQ domain, combined with structure-based molecular simulation of corresponding CaM/IQ binding energy perturbations, suggests that the C lobe of CaM partially dislodges from the IQ element during channel regulation, allowing exposed IQ residues to trigger regulation via isoform-specific interactions with alternative channel regions.
Coarse-Grained MD Simulations of Membrane Protein-Bilayer Self-Assembly by Kathryn A. Scott; Peter J. Bond; Anthony Ivetac; Alan P. Chetwynd; Syma Khalid; Mark S.P. Sansom (621-630).
Complete determination of a membrane protein structure requires knowledge of the protein position within the lipid bilayer. As the number of determined structures of membrane proteins increases so does the need for computational methods which predict their position in the lipid bilayer. Here we present a coarse-grained molecular dynamics approach to lipid bilayer self-assembly around membrane proteins. We demonstrate that this method can be used to predict accurately the protein position in the bilayer for membrane proteins with a range of different sizes and architectures.
Infinite Kinetic Stability against Dissociation of Supramolecular Protein Complexes through Donor Strand Complementation by Chasper Puorger; Oliv Eidam; Guido Capitani; Denis Erilov; Markus G. Grütter; Rudi Glockshuber (631-642).
Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through “donor strand complementation,” whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 × 109 years. The 15 residue peptide forms ideal intermolecular β sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.
Structural Basis of Site-Specific Histone Recognition by the Bromodomains of Human Coactivators PCAF and CBP/p300 by Lei Zeng; Qiang Zhang; Guillermo Gerona-Navarro; Natalia Moshkina; Ming-Ming Zhou (643-652).
Histone lysine acetylation is central to epigenetic control of gene transcription. Bromodomains of chromosomal proteins function as acetyl-lysine (Kac) binding domains. However, how bromodomains recognize site-specific histones remains unanswered. Here, we report three three-dimensional solution structures of the bromodomains of the human transcriptional coactivators CREB-binding protein (CBP) and p300/CBP-associated factor (PCAF) bound to peptides derived from histone acetylation sites at lysines 36 and 9 in H3, and lysine 20 in H4. From structural and biochemical binding analyses, we determine consensus histone recognition by the bromodomains of PCAF and CBP, which represent two different subgroups of the bromodomain family. Through bromodomain residues in the ZA and BC loops, PCAF prefers acetylation sites with a hydrophobic residue at (Kac+2) position and a positively charged or aromatic residue at (Kac+3), whereas CBP favors bulky hydrophobic residues at (Kac+1) and (Kac+2), a positively charged residue at (Kac-1), and an aromatic residue at (Kac-2).
Keywords: PROTEINS; DNA;