Structure (v.19, #4)

In This Issue (v-vi).

Membrane Protein Structure Determination using Paramagnetic Tags by Soumya Ganguly; Brian E. Weiner; Jens Meiler (441-443).
The combination of paramagnetic tagging strategies with NMR or EPR spectroscopic techniques can revolutionize de novo structure determination of helical membrane proteins. Leveraging the full potential of this approach requires optimal labeling strategies and prediction of membrane protein topology from sparse and low-resolution distance restraints, as addressed by .

Notch activation requires unfolding of a juxtamembrane negative regulatory domain (NRR). analyzed the dynamics of NRR unfolding in the presence of EGTA. As predicted from the crystal structure and deletion analyses, the lin-Notch repeats unfold first, facilitating access by ADAM proteases. Surprisingly, the heterodimerization domain remains stable.

Closing In on the Hsp90 Chaperone-Client Relationship by Klaus Richter; Johannes Buchner (445-446).
The molecular chaperone Hsp90 regulates the activity and stability of a set of client proteins. Despite progress in understanding its mechanism, the interaction of Hsp90 with clients has remained enigmatic. Now, in a recent issue of Molecular Cell, Street and coworkers present results that integrate the client in the Hsp90 chaperone cycle.

The Structural Biology of Toll-like Receptors by Istvan Botos; David M. Segal; David R. Davies (447-459).
The membrane-bound Toll-like receptors (TLRs) trigger innate immune responses after recognition of a wide variety of pathogen-derived compounds. Despite the wide range of ligands recognized by TLRs, the receptors share a common structural framework in their extracellular, ligand-binding domains. These domains all adopt horseshoe-shaped structures built from leucine-rich repeat motifs. Typically, on ligand binding, two extracellular domains form an “m”-shaped dimer sandwiching the ligand molecule bringing the transmembrane and cytoplasmic domains in close proximity and triggering a downstream signaling cascade. Although the ligand-induced dimerization of these receptors has many common features, the nature of the interactions of the TLR extracellular domains with their ligands varies markedly between TLR paralogs.► TLRs recognize pathogen-derived ligands by their ectodomains ► TLR ECDs are horseshoe-shaped structures built from leucine-rich repeats ► TLR-ligands dimerize ectodomains via their lateral faces, forming “m”-shaped structures ► Dimerization leads to downstream signaling whose structural basis is still unknown

The thioredoxin family of oxidoreductases plays an important role in redox signaling and control of protein function. Not only are thioredoxins linked to a variety of disorders, but their stable structure has also seen application in protein engineering. Both sequence-based and structure-based tools exist for thioredoxin identification, but remote homolog detection remains a challenge. We developed a thioredoxin predictor using the approach of integrating sequence with structural information. We combined a sequence-based Hidden Markov Model (HMM) with a molecular dynamics enhanced structure-based recognition method (dynamic FEATURE, DF). This hybrid method (HMMDF) has high precision and recall (0.90 and 0.95, respectively) compared with HMM (0.92 and 0.87, respectively) and DF (0.82 and 0.97, respectively). Dynamic FEATURE is sensitive but struggles to resolve closely related protein families, while HMM identifies these evolutionary differences by compromising sensitivity. Our method applied to structural genomics targets makes a strong prediction of a novel thioredoxin.Display Omitted► New structure-based thioredoxin model allows more exact active site representation ► MD simulation improves recall of structure-based function prediction methods ► Implicit solvent MD simulation is sufficiently precise for simulating thioredoxins ► Sequence and structural dynamics data jointly improve remote thioredoxin detection

Crystal Structure of Type III Glutamine Synthetase: Surprising Reversal of the Inter-Ring Interface by Jason M. van Rooyen; Valerie R. Abratt; Hassan Belrhali; Trevor Sewell (471-483).
Glutamine synthetases are ubiquitous, homo-oligomeric enzymes essential for nitrogen metabolism. Unlike types I and II, which are well described both structurally and functionally, the larger, type IIIs are poorly characterized despite their widespread occurrence. An understanding of the structural basis for this divergence and the implications for design of type-specific inhibitors has, therefore, been impossible. The first crystal structure of a GSIII enzyme, presented here, reveals a conservation of the GS catalytic fold but subtle differences in protein-ligand interactions suggest possible avenues for the design GSIII inhibitors. Despite these similarities, the divergence of the GSIII enzymes can be explained by differences in quaternary structure. Unexpectedly, the two hexameric rings of the GSIII dodecamer associate on the opposite surface relative to types I and II. The diversity of GS quaternary structures revealed here suggests a nonallosteric role for the evolution of the double-ringed architecture seen in all GS enzymes.Display Omitted► The divergence of the GSIIIs is reflected in differences in quaternary structure ► Nonallosteric origin for conserved double-ring GS architectures ► Fold and active site is conserved in the GSIIIs despite divergence ► Subtle differences in the GSIII active site may be exploitable for drug design

Optimal Mutation Sites for PRE Data Collection and Membrane Protein Structure Prediction by Huiling Chen; Fei Ji; Victor Olman; Charles K. Mobley; Yizhou Liu; Yunpeng Zhou; John H. Bushweller; James H. Prestegard; Ying Xu (484-495).
Nuclear magnetic resonance paramagnetic relaxation enhancement (PRE) measures long-range distances to isotopically labeled residues, providing useful constraints for protein structure prediction. The method usually requires labor-intensive conjugation of nitroxide labels to multiple locations on the protein, one at a time. Here a computational procedure, based on protein sequence and simple secondary structure models, is presented to facilitate optimal placement of a minimum number of labels needed to determine the correct topology of a helical transmembrane protein. Tests on DsbB (four helices) using just one label lead to correct topology predictions in four of five cases, with the predicted structures <6 Å to the native structure. Benchmark results using simulated PRE data show that we can generally predict the correct topology for five and six to seven helices using two and three labels, respectively, with an average success rate of 76% and structures of similar precision. The results show promise in facilitating experimentally constrained structure prediction of membrane proteins.► A method to guide optimal PRE data collection for membrane proteins is illustrated ► Optimal sites are predicted with high accuracy from sequence information alone ► Promises to facilitate NMR protein structure prediction of larger membrane proteins

Peering Down the Barrel of a Bacteriophage Portal: The Genome Packaging and Release Valve in P22 by Jinghua Tang; Gabriel C. Lander; Adam Olia; Rui Li; Sherwood Casjens; Peter Prevelige; Gino Cingolani; Timothy S. Baker; John E. Johnson (496-502).
The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 Å resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by were fitted into this reconstruction. The interpreted density map revealed that the 150 Å, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins.► Bacteriophage P22 packages its dsDNA genome in ordered layers at liquid-crystalline density ► DNA remains within the capsid during the addition of tail machine closure proteins ► The 12, C-terminal, 130 residue polypeptides of the portal forms a 200 Å long “barrel” ► The barrel functions as a “Chinese finger trap” to transiently retain the DNA

The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor by Tzu-Lan Yeh; Chih-Yung S. Lee; L. Mario Amzel; Peter J. Espenshade; Mario A. Bianchet (503-514).
Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 Å resolution shows an all-α-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.Display Omitted► The crystal structure shows that Nro1 is a HEAT-repeat motif protein ► Nro1 is the nuclear import adaptor for Ofd1 ► Kap123, a karyopherin-β, mediates the nuclear import of the Nro1-Ofd1 complex ► Inhibition and nuclear localization of Ofd1 by Nro1 map to its N-terminal α helix

Structural Insights into the Architecture and Allostery of Full-Length AMP-Activated Protein Kinase by Li Zhu; Lei Chen; Xiao-Ming Zhou; Yuan-Yuan Zhang; Yi-Jiong Zhang; Jing Zhao; Shang-Rong Ji; Jia-Wei Wu; Yi Wu (515-522).
AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of α catalytic subunit, β scaffolding subunit, and γ regulatory subunit with critical roles in maintaining cellular energy homeostasis. However, the molecular architecture of the intact complex and the allostery associated with the adenosine binding-induced regulation of kinase activity remain unclear. Here, we determine the three-dimensional reconstruction and subunit organization of the full-length rat AMPK (α1β1γ1) through single-particle electron-microscopy. By comparing the structures of AMPK in ATP- and AMP-bound states, we are able to visualize the sequential conformational changes underlying kinase activation that transmits from the adenosine binding sites in the γ subunit to the kinase domain of the α subunit. These results not only make substantial revision to the current model of AMPK assembly, but also highlight a central role of the linker sequence of the α subunit in mediating the allostery of AMPK.Display Omitted► The molecular architecture of full-length AMPK is determined by single-particle EM ► Sequential conformational changes underlying AMPK activation are observed ► A three-state model for AMPK structure and regulation is proposed ► A central role of the linker sequence of α subunit in AMPK allostery is highlighted

Reintroducing Electrostatics into Macromolecular Crystallographic Refinement: Application to Neutron Crystallography and DNA Hydration by Timothy D. Fenn; Michael J. Schnieders; Marat Mustyakimov; Chuanjie Wu; Paul Langan; Vijay S. Pande; Axel T. Brunger (523-533).
Most current crystallographic structure refinements augment the diffraction data with a priori information consisting of bond, angle, dihedral, planarity restraints, and atomic repulsion based on the Pauli exclusion principle. Yet, electrostatics and van der Waals attraction are physical forces that provide additional a priori information. Here, we assess the inclusion of electrostatics for the force field used for all-atom (including hydrogen) joint neutron/X-ray refinement. Two DNA and a protein crystal structure were refined against joint neutron/X-ray diffraction data sets using force fields without electrostatics or with electrostatics. Hydrogen-bond orientation/geometry favors the inclusion of electrostatics. Refinement of Z-DNA with electrostatics leads to a hypothesis for the entropic stabilization of Z-DNA that may partly explain the thermodynamics of converting the B form of DNA to its Z form. Thus, inclusion of electrostatics assists joint neutron/X-ray refinements, especially for placing and orienting hydrogen atoms.► Electrostatics improve water molecule bonding and geometry in crystallographic refinement ► Electrostatics improves agreement with neutron data ► AMOEBA with PME has advantages over fixed charges with spherical cutoffs ► The spine of hydration in Z-DNA is disordered

Locked Tether Formation by Cooperative Folding of Rna14p Monkeytail and Rna15p Hinge Domains in the Yeast CF IA Complex by Maria Moreno-Morcillo; Lionel Minvielle-Sébastia; Sébastien Fribourg; Cameron D. Mackereth (534-545).
The removal of the 3′ region of pre-mRNA followed by polyadenylation is a key step in mRNA maturation. In the yeast Saccharomyces cerevisiae, one component of the processing machinery is the cleavage/polyadenylation factor IA (CF IA) complex, composed of four proteins (Clp1p, Pcf11p, Rna14p, Rna15p) that recognize RNA sequences adjacent to the cleavage site and recruit additional processing factors. To gain insight into the molecular architecture of CF IA we solved the solution structure of the heterodimer composed of the interacting regions between Rna14p and Rna15p. The C-terminal monkeytail domain from Rna14p and the hinge region from Rna15p display a coupled binding and folding mechanism, where both peptides are initially disordered. Mutants with destabilized monkeytail-hinge interactions prevent association of Rna15p within CF IA. Conservation of interdomain residues reveals that the structural tethering is preserved in the homologous mammalian cleavage stimulation factor (CstF)-77 and CstF-64 proteins of the CstF complex.Display Omitted► Tight association by Rna15p hinge region with newly identified Rna14p monkeytail ► Bimolecular structure formed by a helical peptide within a second helical domain ► Known mutations target monkeytail or hinge regions and release Rna15p from CF IA ► Conservation of buried residues predict similar tether between CstF-77 and CstF-64

Evidence for Increased Exposure of the Notch1 Metalloprotease Cleavage Site upon Conversion to an Activated Conformation by Kittichoat Tiyanont; Thomas E. Wales; Miguel Aste-Amezaga; Jon C. Aster; John R. Engen; Stephen C. Blacklow (546-554).
Notch proteins are transmembrane receptors that normally adopt a resting state poised to undergo activating proteolysis upon ligand engagement. Receptor quiescence is maintained by three LIN12/Notch repeats (LNRs), which wrap around a heterodimerization domain (HD) divided by furin cleavage at site S1 during maturation. Ligand binding initiates signaling by inducing sensitivity of the HD to proteolysis at the regulated S2 cleavage site. Here, we used hydrogen exchange mass spectrometry to examine the solution dynamics of the Notch1 negative regulatory region in autoinhibited states before and after S1 cleavage, in a proteolytically sensitive “on” state, and in a complex with an inhibitory antibody. Conversion to the “on” state leads to accelerated deuteration in the S2 region and in nearby secondary structural elements within the HD. In contrast, complexation with the inhibitory antibody retards deuteration around the S2 site. Together, these studies reveal how S2 site exposure is promoted by receptor activation and suppressed by inhibitory antibodies.Display Omitted► First use of HX-MS to investigate the dynamics of the Notch activation switch ► In the autoinhibited conformation, the S2 site is protected against exchange ► The dynamics of the protein are unaffected upon maturation cleavage by furin at S1 ► Conversion to the “on” state leads to accelerated deuteration of the S2 site

Binding-induced backbone and large-scale conformational changes represent one of the major challenges in the modeling of biomolecular complexes by docking. To address this challenge, we have developed a flexible multidomain docking protocol that follows a “divide-and-conquer” approach to model both large-scale domain motions and small- to medium-scale interfacial rearrangements: the flexible binding partner is treated as an assembly of subparts/domains that are docked simultaneously making use of HADDOCK's multidomain docking ability. For this, the flexible molecules are cut at hinge regions predicted using an elastic network model. The performance of this approach is demonstrated on a benchmark covering an unprecedented range of conformational changes of 1.5 to 19.5 Å. We show from a statistical survey of known complexes that the cumulative sum of eigenvalues obtained from the elastic network has some predictive power to indicate the extent of the conformational change to be expected.Display Omitted► A new HADDOCKing protocol to address large-scale domain rearrangements ► This divide-and-conquer approach uses HADDOCK's multidomain docking ability ► Methodology to model conformational changes as large as 19.5 Å ► Indicators are provided to predict possible large conformational changes

Beyond the Random Coil: Stochastic Conformational Switching in Intrinsically Disordered Proteins by Ucheor B. Choi; James J. McCann; Keith R. Weninger; Mark E. Bowen (566-576).
Intrinsically disordered proteins (IDPs) participate in critical cellular functions that exploit the flexibility and rapid conformational fluctuations of their native state. Limited information about the native state of IDPs can be gained by the averaging over many heterogeneous molecules that is unavoidable in ensemble approaches. We used single molecule fluorescence to characterize native state conformational dynamics in five synaptic proteins confirmed to be disordered by other techniques. For three of the proteins, SNAP-25, synaptobrevin and complexin, their conformational dynamics could be described with a simple semiflexible polymer model. Surprisingly, two proteins, neuroligin and the NMDAR-2B glutamate receptor, were observed to stochastically switch among distinct conformational states despite the fact that they appeared intrinsically disordered by other measures. The hop-like intramolecular diffusion found in these proteins is suggested to define a class of functionality previously unrecognized for IDPs.► Not all intrinsically disordered proteins can be described by simple polymer models ► Stochastic conformational switching in neuroligin is a new form of protein structure ► Membrane reconstitution did not alter conformational dynamics in synaptobrevin ► SNARE binding increases the volume of the disordered C-terminal domain of complexin

Structural Insights into Rcs Phosphotransfer: The Newly Identified RcsD-ABL Domain Enhances Interaction with the Response Regulator RcsB by Kerstin Schmöe; Vladimir V. Rogov; Natalia Yu. Rogova; Frank Löhr; Peter Güntert; Frank Bernhard; Volker Dötsch (577-587).
The Rcs-signaling system is one of the most remarkable phosphorelay pathways in Enterobacteriaceae, comprising several membrane-bound and soluble proteins. Within the complex phosphotransfer pathway, the histidine phosphotransferase (HPt) domain of the RcsD membrane-bound component serves as a crucial factor in modulating the phosphorylation state of the transcription factor RcsB. We have identified a new domain, RcsD-ABL, located N terminally to RcsD-HPt that interacts with RcsB as well. We have determined its structure, characterized its interaction interface with RcsB, and built a structural model of the complex of the RcsD-ABL domain with RcsB. Our results indicate that the effector domain of RcsB, which normally binds to DNA, is recognized by RcsD-ABL, whereas the HPt domain interacts with the phosphoreceiver domain of RcsB.► NMR structure of a newly identified domain within RcsD named RcsD-ABL ► RcsD-ABL functions as a binding domain for the response regulator RcsB ► Mapping the interface of the protein complex RcsD-ABL:RcsB ► Newly identified domain stabilizes binding of RcsB to RcsD-HPt

F1-ATPase, a rotary motor powered by adenosine triphosphate hydrolysis, has been extensively studied by various methods. Here, we performed a systematic comparison of 29 X-ray crystal structures of F1-complexes, finding fine interplay among enzyme structures, catalysis, and rotations. First, analyzing the 87 structures of enzymatic αβ-subunits, we confirmed that the two modes, the hinge motion of β-subunit and the loose/tight motion of the αβ-interface, dominate the variations. The structural ensemble was nearly contiguous bridging three clusters, αβTP, αβDP, and αβE. Second, the catalytic site analysis suggested the correlation between the phosphate binding and the tightening of the αβ-interface. Third, addressing correlations of enzymatic structures with the orientations of the central stalk γ, we found that the γ rotation highly correlates with loosening of αβE-interface and βDP hinge motions. Finally, calculating the helix 6 angle of β, we identified the recently observed partially closed conformation being consistent with βHC.► Structural comparison of 87 αβ enzyme structures and catalytic sites was performed ► Rotary and tilting angles of γ were calculated for 29 F1-complexes ► We found γ-rotation highly correlates with αβE-interface and βDP hinge structures ► From modeling of helix6, the partially closed conformation may correspond to βHC