Structure (v.16, #1)

The Soul of a New Structure-Function Machine by Sylvie Doublié; Mark A. Rould (3-4).

Contributions to the NIH-NIGMS Protein Structure Initiative from the PSI Production Centers by Stephen K. Burley; Andrzej Joachimiak; Gaetano T. Montelione; Ian A. Wilson (5-11).

Harnessing Knowledge from Structural Genomics by Helen M. Berman (16-18).

A Nice Return on the “Stalk” Exchange by Lori L. Burrows (19-20).
Type IV Pili (T4P) are protein filaments widely used by bacterial pathogens for adherence and motility. In this issue of Structure, Li and colleagues made clever use of hydrogen/deuterium exchange mass spectrometry techniques to reveal surprising insights into the ultrastructure of T4P.

A Proposed Time-Resolved X-Ray Scattering Approach to Track Local and Global Conformational Changes in Membrane Transport Proteins by Magnus Andersson; Jonathan Vincent; David van der Spoel; Jan Davidsson; Richard Neutze (21-28).
Time-resolved X-ray scattering has emerged as a powerful technique for studying the rapid structural dynamics of small molecules in solution. Membrane-protein-catalyzed transport processes frequently couple large-scale conformational changes of the transporter with local structural changes perturbing the uptake and release of the transported substrate. Using light-driven halide ion transport catalyzed by halorhodopsin as a model system, we combine molecular dynamics simulations with X-ray scattering calculations to demonstrate how small-molecule time-resolved X-ray scattering can be extended to the study of membrane transport processes. In particular, by introducing strongly scattering atoms to label specific positions within the protein and substrate, the technique of time-resolved wide-angle X-ray scattering can reveal both local and global conformational changes. This approach simultaneously enables the direct visualization of global rearrangements and substrate movement, crucial concepts that underpin the alternating access paradigm for membrane transport proteins.
Keywords: PROTEIN;

Structural Insights into the Multifunctional Protein VP3 of Birnaviruses by Arnau Casañas; Aitor Navarro; Cristina Ferrer-Orta; Dolores González; José F. Rodríguez; Núria Verdaguer (29-37).
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most harmful poultry diseases. The IBDV genome encodes five mature proteins; of these, the multifunctional protein VP3 plays an essential role in virus morphogenesis. This protein, which interacts with the structural protein VP2, with the double-stranded RNA genome, and with the virus-encoded, RNA-dependent RNA polymerase, VP1, is involved not only in the formation of the viral capsid, but also in the recruitment of VP1 into the capsid and in the encapsidation of the viral genome. Here, we report the X-ray structure of the central region of VP3, residues 92–220, consisting of two α-helical domains connected by a long and flexible hinge that are organized as a dimer. Unexpectedly, the overall fold of the second VP3 domain shows significant structural similarities with different transcription regulation factors.

Serpins display a number of highly unusual structural properties along with a unique mechanism of inhibition. Although structures of numerous serpins have been solved by X-ray crystallography, little is known about the dynamics of serpins in their inhibitory active conformation. In this study, two complementary structural mass spectrometry methods, hydroxyl radical-mediated footprinting and hydrogen/deuterium (H/D) exchange, were employed to highlight differences between the static crystal structure and the dynamic conformation of human serpin protein, α1-antitrypsin (α1AT). H/D exchange revealed the distribution of flexible and rigid regions of α1AT, whereas footprinting revealed the dynamic environments of several side chains previously identified as important for the metastability of α1AT. This work provides insights into the unique structural design of α1AT and improves our understanding of its unusual inhibition mechanism. Also, we demonstrate that the combination of the two MS techniques provides a more complete picture of protein structure than either technique alone.
Keywords: PROTEINS;

Structure of the Yeast tRNA m7G Methylation Complex by Nicolas Leulliot; Maxime Chaillet; Dominique Durand; Nathalie Ulryck; Karine Blondeau; Herman van Tilbeurgh (52-61).
Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNAPhe has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.
Keywords: RNA;

Force Generation in Kinesin Hinges on Cover-Neck Bundle Formation by Wonmuk Hwang; Matthew J. Lang; Martin Karplus (62-71).
In kinesin motors, a fundamental question concerns the mechanism by which ATP binding generates the force required for walking. Analysis of available structures combined with molecular dynamics simulations demonstrates that the conformational change of the neck linker involves the nine-residue-long N-terminal region, the cover strand, as an element that is essential for force generation. Upon ATP binding, it forms a β sheet with the neck linker, the cover-neck bundle, which induces the forward motion of the neck linker, followed by a latch-type binding to the motor head. The estimated stall force and anisotropic response to external loads calculated from the model agree with force-clamp measurements. The proposed mechanism for force generation by the cover-neck bundle formation appears to apply to several kinesin families. It also elucidates the design principle of kinesin as the smallest known processive motor.
Keywords: CELLBIO;

The GLA domain, a common membrane-anchoring domain of several serine protease coagulation factors, is a key element in membrane association and activation of these factors in a highly Ca2+-dependent manner. However, the critical role of Ca2+ ions in binding is only poorly understood. Here, we present the atomic model of a membrane-bound GLA domain by using MD simulations of the GLA domain of human factor VIIa and an anionic lipid bilayer. The binding is furnished through a complete insertion of the ω-loop into the membrane and through direct interactions of structurally bound Ca2+ ions and protein side chains with negative lipids. The model suggests that Ca2+ ions play two distinct roles in the process: the four inner Ca2+ ions are primarily responsible for optimal folding of the GLA domain for membrane insertion, whereas the outer Ca2+ ions anchor the protein to the membrane through direct contacts with lipids.

Structures of the Human Orotidine-5′-Monophosphate Decarboxylase Support a Covalent Mechanism and Provide a Framework for Drug Design by Julia G. Wittmann; Daniel Heinrich; Kathrin Gasow; Alexandra Frey; Ulf Diederichsen; Markus G. Rudolph (82-92).
UMP synthase (UMPS) catalyzes the last two steps of de novo pyrimidine nucleotide synthesis and is a potential cancer drug target. The C-terminal domain of UMPS is orotidine-5′-monophosphate decarboxylase (OMPD), a cofactor-less yet extremely efficient enzyme. Studies of OMPDs from micro-organisms led to the proposal of several noncovalent decarboxylation mechanisms via high-energy intermediates. We describe nine crystal structures of human OMPD in complex with substrate, product, and nucleotide inhibitors. Unexpectedly, simple compounds can replace the natural nucleotides and induce a closed conformation of OMPD, defining a tripartite catalytic site. The structures outline the requirements drugs must meet to maximize therapeutic effects and minimize cross-species activity. Chemical mimicry by iodide identified a CO2 product binding site. Plasticity of catalytic residues and a covalent OMPD-UMP complex prompt a reevaluation of the prevailing decarboxylation mechanism in favor of covalent intermediates. This mechanism can also explain the observed catalytic promiscuity of OMPD.
Keywords: PROTEINS;

Extended Polypeptide Linkers Establish the Spatial Architecture of a Pyruvate Dehydrogenase Multienzyme Complex by Jeffrey S. Lengyel; Katherine M. Stott; Xiongwu Wu; Bernard R. Brooks; Andrea Balbo; Peter Schuck; Richard N. Perham; Sriram Subramaniam; Jacqueline L.S. Milne (93-103).
Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of ∼75–90 Å from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.
Keywords: PROTEINS;

Structures of the Human Pyruvate Dehydrogenase Complex Cores: A Highly Conserved Catalytic Center with Flexible N-Terminal Domains by Xuekui Yu; Yasuaki Hiromasa; Hua Tsen; James K. Stoops; Thomas E. Roche; Z. Hong Zhou (104-114).
Dihydrolipoyl acetyltransferase (E2) is the central component of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA. Structural comparison by cryo-electron microscopy (cryo-EM) of the human full-length and truncated E2 (tE2) cores revealed flexible linkers emanating from the edges of trimers of the internal catalytic domains. Using the secondary structure constraints revealed in our 8 Å cryo-EM reconstruction and the prokaryotic tE2 atomic structure as a template, we derived a pseudo atomic model of human tE2. The active sites are conserved between prokaryotic tE2 and human tE2. However, marked structural differences are apparent in the hairpin domain and in the N-terminal helix connected to the flexible linker. These permutations away from the catalytic center likely impart structures needed to integrate a second component into the inner core and provide a sturdy base for the linker that holds the pyruvate dehydrogenase for access by the E2-bound regulatory kinase/phosphatase components in humans.
Keywords: PROTEINS;

Structure of the Human Protein Kinase MPSK1 Reveals an Atypical Activation Loop Architecture by Jeyanthy Eswaran; Antonio Bernad; Jose M. Ligos; Barbara Guinea; Judit É. Debreczeni; Frank Sobott; Sirlester A. Parker; Rafael Najmanovich; Benjamin E. Turk; Stefan Knapp (115-124).
The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a β sheet and a large α-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-ϕ-H/Y-T-N/G-X-X-X (ϕ is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

Roles of Structure and Structural Dynamics in the Antibody Recognition of the Allergen Proteins: An NMR Study on Blomia tropicalis Major Allergen by Mandar T. Naik; Chi-Fon Chang; I.-Chun Kuo; Camy C.-H. Kung; Fong-Cheng Yi; Kaw-Yan Chua; Tai-Huang Huang (125-136).
Blo t 5 is the major allergen from Blomia tropicalis mites and shows strong IgE reactivity with up to 90% of asthmatic and rhinitis patients' sera. The NMR solution structure of Blo t 5 comprises three long α helices, forming a coiled-coil, triple-helical bundle with a left-handed twist. TROSY-NMR experiments were used to study Blo t 5 interaction with the Fab′ of a specific monoclonal antibody, mAb 4A7. The mAb epitope comprises two closely spaced surfaces, I and II, connected by a sharp turn and bearing critical residues Asn46 and Lys47 on one surface, and Lys54 and Arg57 on the other. This discontinuous epitope overlaps with the human IgE epitope(s) of Blo t 5. Epitope surface II is further predicted to undergo conformational exchange in the millisecond to microsecond range. The results presented are critical for the design of a hypoallergenic Blo t 5 for efficacious immunotherapy of allergic diseases.

Vibrio cholerae Toxin-Coregulated Pilus Structure Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry by Juliana Li; Mindy S. Lim; Sheng Li; Melissa Brock; Michael E. Pique; Virgil L. Woods; Lisa Craig (137-148).
The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show that the pilin subunits are held together by tight packing of the N-terminal α helices, but loose packing of the C-terminal globular domains leaves substantial gaps on the filament surface. These gaps expose a glycine-rich, amphipathic segment of the N-terminal α-helix, contradicting the consensus view that this region is buried in the filament core. Our results explain extreme filament flexibility, suggest a molecular basis for pilus-pilus interactions, and reveal a previously unrecognized therapeutic target for V. cholerae and other enteric pathogens.

Structure and DNA Binding of the Human Rtf1 Plus3 Domain by Rob N. de Jong; Vincent Truffault; Tammo Diercks; Eiso AB; Mark A. Daniels; Rob Kaptein; Gert E. Folkers (149-159).
The yeast Paf1 complex consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and regulates histone H2B ubiquitination, histone H3 methylation, RNA polymerase II carboxy-terminal domain (CTD) Ser2 phosphorylation, and RNA 3′ end processing. We provide structural insight into the Paf1 complex with the NMR structure of the conserved and functionally important Plus3 domain of human Rtf1. A predominantly β-stranded subdomain displays structural similarity to Dicer/Argonaute PAZ domains and to Tudor domains. We further demonstrate that the highly basic Rtf1 Plus3 domain can interact in vitro with single-stranded DNA via residues on the rim of the β sheet, reminiscent of siRNA binding by PAZ domains, but did not detect binding to double-stranded DNA or RNA. We discuss the potential role of Rtf1 Plus3 ssDNA binding during transcription elongation.
Keywords: DNA; RNA;