Structure (v.18, #4)

In This Issue (v-vi).

Recent computational studies () have brought us closer to watching a molecular machine, myosin, in action. This significant achievement, obtained by sensible physical approximations, opens a highway to improve the integration of computational methods and experimental results.

In this issue, provided interesting insights concerning the molecular details of how the meandering of disordered proteins in conformational space can lead to collective binding modes and ultrasensitive probing of cellular kinase activities.

And Yet It Moves: Active Site Remodeling in the SUMO E1 by Daniel Völler; Hermann Schindelin (419-421).
The activation of ubiquitin and ubiquin-like proteins is catalyzed by E1 enzymes via consecutive adenylation and thioesterification reactions involving two apparently distant active sites. uncover dramatic conformational changes in the SUMO E1 that spatially merge the adenylation and thioesterification active sites, including a > 30 Å movement of the active site cysteine.

Dynameomics: A Comprehensive Database of Protein Dynamics by Marc W. van der Kamp; R. Dustin Schaeffer; Amanda L. Jonsson; Alexander D. Scouras; Andrew M. Simms; Rudesh D. Toofanny; Noah C. Benson; Peter C. Anderson; Eric D. Merkley; Steven Rysavy; Dennis Bromley; David A.C. Beck; Valerie Daggett (423-435).
Display Omitted
Keywords: PROTEINS;

Structure of Concatenated HAMP Domains Provides a Mechanism for Signal Transduction by Michael V. Airola; Kylie J. Watts; Alexandrine M. Bilwes; Brian R. Crane (436-448).
HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.► Presents the first poly-HAMP structure and identifies a novel HAMP domain conformation ► HAMP conformations differ by changes in helical register, rotation, and crossing angle ► Proposes a new signal transduction model consistent with known signal inputs to HAMP ► Provides an output mechanism for HAMP domains involving stutter compensation

RNA 3′-phosphate cyclase (RtcA) synthesizes RNA 2′,3′ cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-AMP intermediate; transfer of adenylate to an RNA 3′-phosphate to form RNA(3′)pp(5′)A; and attack of the vicinal O2′ on the 3′-phosphorus to form a 2′,3′ cyclic phosphate. Here we report the 1.7 Å crystal structure of the RtcA-AMP intermediate, which reveals the mechanism of nucleotidyl transfer. Adenylate is linked via a phosphoamide bond to the His309 Nɛ atom. A network of hydrogen bonds to the ribose O2′ and O3′ accounts for the stringent ribonucleotide preference. Adenine is sandwiched in a hydrophobic pocket between Tyr284 and Pro131 and the preference for adenine is enforced by Phe135, which packs against the purine C2 edge. Two sulfates bound near the adenylate plausibly mimic the 3′-terminal and penultimate phosphates of RNA. The structure illuminates how the four α2/β4 domains contribute to substrate binding and catalysis.► RNA 2′,3′ cyclic phosphate ends play important roles in RNA metabolism ► RNA 3′-terminal phosphate cyclase (Rtc) enzymes catalyze de novo cyclization ► Cyclization entails formation of a covalent enzyme-(histidinyl-Nɛ)-AMP intermediate ► The 1.7 Å crystal structure of Rtc-AMP reveals the mechanism of nucleotidyl transfer
Keywords: PROTEINS;

Pi Release from Myosin: A Simulation Analysis of Possible Pathways by Marco Cecchini; Yuri Alexeev; Martin Karplus (458-470).
The release of phosphate (Pi) is an important element in actomyosin function and has been shown to be accelerated by the binding of myosin to actin. To provide information about the structural elements important for Pi release, possible escape pathways from various isolated myosin II structures have been determined by molecular dynamics simulations designed for studying such slow processes. The residues forming the pathways were identified and their role was evaluated by mutant simulations. Pi release is slow in the pre-powerstroke structure, an important element in preventing the powerstroke prior to actin binding, and is much more rapid for Pi modeled into the post-rigor and rigor-like structures. The previously proposed backdoor route is dominant in the pre-powerstroke and post-rigor states, whereas a different path is most important in the rigor-like state. This finding suggests a mechanism for the actin-activated acceleration of Pi release.Display Omitted► Pathways for Pi release from myosin II are determined at atomic resolution ► The escape barrier is compared in various myosin II structures ► The origin of the high-release barrier in the myosin pre-powerstroke is described ► A novel mechanism for accelerated Pi release from actomyosin is proposed

The detachment kinetics from actin upon ATP binding is a key step in the reaction cycle of myosin V. We show that a network of residues, constituting the allostery wiring diagram (AWD), that trigger the rigor (R) to post-rigor (PR) transition, span key structural elements from the ATP and actin-binding regions. Several of the residues are in the 33 residue helix (H18), P loop, and switch I. Brownian dynamics simulations show that a hierarchy of kinetically controlled local structural changes leads to the opening of the “cleft” region, resulting in the detachment of the motor domain from actin. Movements in switch I and P loop facilitate changes in the rest of the motor domain, in particular the rotation of H18, whose stiffness within the motor domain is crucial in the R → PR transition. The finding that residues in the AWD also drive the kinetics of the R → PR transition shows how the myosin architecture regulates the allosteric movements during the reaction cycle.► A signaling network of residues describes myosin V rigor to post-rigor transition ► Hierarchy of movements of a few structural elements drives the R to PR transition ► Dynamics of the R to PR transition is encoded by the architecture of myosin motor ► Proposed theoretical methods are applicable to describe motility in molecular motors

The Human Breast Cancer Resistance Protein (BCRP/ABCG2) Shows Conformational Changes with Mitoxantrone by Mark F. Rosenberg; Zsolt Bikadi; Janice Chan; Xiaoping Liu; Zhanglin Ni; Xiaokun Cai; Robert C. Ford; Qingcheng Mao (482-493).
BCRP/ABCG2 mediates efflux of drugs and xenobiotics. BCRP was expressed in Pichia pastoris, purified to > 90% homogeneity, and subjected to two-dimensional (2D) crystallization. The 2D crystals showed a p121 symmetry and projection maps were determined at 5 Å resolution by cryo-electron microscopy. Two crystal forms with and without mitoxantrone were observed with unit cell dimensions of a = 55.4 Å, b = 81.4 Å, γ = 89.8°, and a = 57.3 Å, b = 88.0 Å, γ = 89.7°, respectively. The projection map without mitoxantrone revealed an asymmetric structure with ring-shaped density features probably corresponding to a bundle of transmembrane α helices, and appeared more open and less symmetric than the map with mitroxantrone. The open and closed inward-facing forms of BCRP were generated by homology modeling, representing the substrate-free and substrate-bound conformations in the absence of nucleotide, respectively. These models are consistent with the experimentally observed conformational change upon substrate binding.► Breast cancer resistance protein (BCRP) belongs to the ABC transporter family ► BCRP is a major cause of chemotherapeutic failure in cancers and leukemia ► Purified BCRP was crystallized in two-dimensions with the drug mitoxantrone ► Projection maps and homology modeling were used to understand conformational changes in the drug transport cycle

Structure/Function Implications in a Dynamic Complex of the Intrinsically Disordered Sic1 with the Cdc4 Subunit of an SCF Ubiquitin Ligase by Tanja Mittag; Joseph Marsh; Alexander Grishaev; Stephen Orlicky; Hong Lin; Frank Sicheri; Mike Tyers; Julie D. Forman-Kay (494-506).
Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.► Intrinsically disordered Sic1 and pSic1 contain significant transient structure ► Structure modulates electrostatic field experienced by folded binding partner Cdc4 ► Free pSic1 ensemble enables calculation of a model of the dynamic Sic1-Cdc4 complex ► Sic1 conformers can reach the catalytic cysteine in ubiquitin ligase complexes

Structural and Dynamical Insights into the Opening Mechanism of P. aeruginosa OprM Channel by Gilles Phan; Houssain Benabdelhak; Marie-Bernard Lascombe; Philippe Benas; Stéphane Rety; Martin Picard; Arnaud Ducruix; Catherine Etchebest; Isabelle Broutin (507-517).
Originally described in bacteria, drug transporters are now recognized as major determinants in antibiotics resistance. For Gram-negative bacteria, the reversible assembly consisting of an inner membrane protein responsible for the active transport, a periplasmic protein, and an exit outer membrane channel achieves transport. The opening of the outer membrane protein OprM from Pseudomonas aeruginosa was modeled through normal mode analysis starting from a new X-ray structure solved at 2.4 Å resolution in P212121 space group. The three monomers are not linked by internal crystallographic symmetries highlighting the possible functional differences. This structure is closed at both ends, but modeling allowed for an opening that is not reduced to the classically proposed “iris-like mechanism.”Display Omitted► A trimer OprM protein structure without three-fold crystallographic axis ► Model of OprM opening by normal mode analysis

Structural Insights into the COP9 Signalosome and Its Common Architecture with the 26S Proteasome Lid and eIF3 by Radoslav I. Enchev; Anne Schreiber; Fabienne Beuron; Edward P. Morris (518-527).
The evolutionary conserved COP9 signalosome (CSN), a large multisubunit complex, plays a central role in regulating ubiquitination and cell signaling. Here we report recombinant insect cell expression and two-step purification of human CSN and demonstrate its functional assembly. We further obtain a three-dimensional structure of both native and recombinant CSN using electron microscopy and single particle analysis. Antibody labeling of CSN5 and segmentation of the structure suggest a likely subunit distribution and the architecture of its helical repeat subunits is revealed. We compare the structure of CSN with its homologous complexes, the 26S proteasome lid and eIF3, and propose a conserved architecture implying similar assembly pathways and/or conserved substrate interaction modes.► Insect cell expression and purification of active human CSN ► The first 3D structure of CSN by electron microscopy at 25 Å resolution ► CSN structure segmentation and labeling of the active site subunit CSN5 ► Structural comparison of homologous PCI complexes: CSN, 26S proteasome lid, and eIF3

Structural Insight into the Sequence Dependence of Nucleosome Positioning by Bin Wu; Kareem Mohideen; Dileep Vasudevan; Curt A. Davey (528-536).
Nucleosome positioning displays sequence dependency and contributes to genomic regulation in a site-specific manner. We solved the structures of nucleosome core particle composed of strong positioning TTTAA elements flanking the nucleosome center. The positioning strength of the super flexible TA dinucleotide is consistent with its observed central location within minor groove inward regions, where it can contribute maximally to energetically challenging minor groove bending, kinking and compression. The marked preference for TTTAA and positioning power of the site 1.5 double helix turns from the nucleosome center relates to a unique histone protein motif at this location, which enforces a sustained, extremely narrow minor groove via a hydrophobic “sugar clamp.” Our analysis sheds light on the basis of nucleosome positioning and indicates that the histone octamer has evolved not to fully minimize sequence discrimination in DNA binding.Display Omitted► TA dinucleotides optimal for histone-imposed minor groove deformation/structure ► Histone-imposed minor groove narrowness is site dependent ► Conserved histone motif yields nucleosome translational positioning mechanism ► Histone octamer has evolved not to fully minimize DNA sequence discrimination
Keywords: DNA; PROTEINS;

Structure of a Virulence Regulatory Factor CvfB Reveals a Novel Winged Helix RNA Binding Module by Yasuhiko Matsumoto; Qingping Xu; Shinya Miyazaki; Chikara Kaito; Carol L. Farr; Herbert L. Axelrod; Hsiu-Ju Chiu; Heath E. Klock; Mark W. Knuth; Mitchell D. Miller; Marc-André Elsliger; Ashley M. Deacon; Adam Godzik; Scott A. Lesley; Kazuhisa Sekimizu; Ian A. Wilson (537-547).
CvfB is a conserved regulatory protein important for the virulence of Staphylococcus aureus. We show here that CvfB binds RNA. The crystal structure of the CvfB ortholog from Streptococcus pneumoniae at 1.4 Å resolution reveals a unique RNA binding protein that is formed from a concatenation of well-known structural modules that bind nucleic acids: three consecutive S1 RNA binding domains and a winged helix (WH) domain. The third S1 and the WH domains are required for cooperative RNA binding and form a continuous surface that likely contributes to the RNA interaction. The WH domain is critical to CvfB function and contains a unique sequence motif. Thus CvfB represents a novel assembly of modules for binding RNA.► CvfB is an RNA binding protein involved in bacterial virulence ► CvfB consists of three consecutive S1 domains and a C-terminal winged helix (WH) domain ► The WH domain and the third S1 domain are involved in cooperative binding of RNA ► WH contains a novel nucleic acid recognition motif