Structure (v.16, #5)
LRR Domain Folding: Just Put a Cap on It!
by Stephanie M.E. Truhlar; Elizabeth A. Komives (pp. 655-657).
In this issue of Structure, describe the role of helical capping motif in nucleating the folding of leucine-rich repeat (LRR) domains.
Telling Bacteria: Do Not LytTR
by Michael Y. Galperin (pp. 657-659).
Transcriptional regulators containing the LytTR-type DNA-binding domain control production of virulence factors in several bacterial pathogens. In this issue of Structure, Sidote et al. report the crystal structure of this elusive domain in complex with its DNA target ().
The Solution to Multiple Structures
by Sophie E. Jackson (pp. 659-661).
Getting high-resolution structures of large proteins in solution has always been a challenge. In this issue of Structure, Krukenberg et al. have used new methods of analyzing SAXS data to reveal a novel conformation of Hsp90 in solution.
Trapping Fugitive Filament Formers
by R. Dyche Mullins (pp. 661-663).
In this issue of Structure, investigate the activation of the Arp2/3 complex and propose a provocative model for Arp2/3-dependent filament formation.
The Mechanics of Translocation: A Molecular “Spring-and-Ratchet” System
by Stephen J. Moran; John F. Flanagan IV; Olivier Namy; David I. Stuart; Ian Brierley; Robert J.C. Gilbert (pp. 664-672).
The translation of genetic information into proteins is a fundamental process of life. Stepwise addition of amino acids to the growing polypeptide chain requires the coordinated movement of mRNA and tRNAs through the ribosome, a process known as translocation. Here, we review current understanding of the kinetics and mechanics of translocation, with particular emphasis on the structure of a functional mammalian ribosome stalled during translocation by an mRNA pseudoknot. In the context of a pseudoknot-stalled complex, the translocase EF-2 is seen to compress a hybrid-state tRNA into a strained conformation. We propose that this strain energy helps overcome the kinetic barrier to translocation and drives tRNA into the P-site, with EF-2 biasing this relaxation in one direction. The tRNA can thus be considered a molecular spring and EF-2 a Brownian ratchet in a “spring-and-ratchet” system within the translocation process.
Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics
by Leonardo G. Trabuco; Elizabeth Villa; Kakoli Mitra; Joachim Frank; Klaus Schulten (pp. 673-683).
A novel method to flexibly fit atomic structures into electron microscopy (EM) maps using molecular dynamics simulations is presented. The simulations incorporate the EM data as an external potential added to the molecular dynamics force field, allowing all internal features present in the EM map to be used in the fitting process, while the model remains fully flexible and stereochemically correct. The molecular dynamics flexible fitting (MDFF) method is validated for available crystal structures of protein and RNA in different conformations; measures to assess and monitor the fitting process are introduced. The MDFF method is then used to obtain high-resolution structures of the E. coli ribosome in different functional states imaged by cryo-EM.
Crystal Structure of NFAT Bound to the HIV-1 LTR Tandem κB Enhancer Element
by Darren L. Bates; Kristen K.B. Barthel; Yongqing Wu; Reza Kalhor; James C. Stroud; Michael J. Giffin; Lin Chen (pp. 684-694).
The host factor, nuclear factor of activated T-cells (NFAT), regulates the transcription and replication of HIV-1. Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem κB enhancer element at 3.05 Å resolution. NFAT binds as a dimer to the upstream κB site (Core II), but as a monomer to the 3′ end of the downstream κB site (Core I). The DNA shows a significant bend near the 5′ end of Core I, where a lysine residue from NFAT bound to the 3′ end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5′ end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for further investigating the functional mechanisms of NFAT in HIV-1 transcription and replication.
Keywords: PROTEINS; DNA
X-Ray Scattering Study of Activated Arp2/3 Complex with Bound Actin-WCA
by Malgorzata Boczkowska; Grzegorz Rebowski; Maxim V. Petoukhov; David B. Hayes; Dmitri I. Svergun; Roberto Dominguez (pp. 695-704).
Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.
The Leucine-Rich Repeat Domain of Internalin B Folds along a Polarized N-Terminal Pathway
by Naomi Courtemanche; Doug Barrick (pp. 705-714).
The leucine-rich repeat domain of Internalin B is composed of seven tandem leucine-rich repeats, which each contain a short β strand connected to a 310 helix by a short turn, and an N-terminal α-helical capping motif. To determine whether folding proceeds along a single, discrete pathway or multiple, parallel pathways, and to map the structure of the transition state ensemble, we examined the effects of destabilizing substitutions of conserved residues in each repeat. We find that, despite the structural redundancy among the repeats, folding proceeds through an N-terminal transition state ensemble in which the extent of structure formation is biased toward repeats one and two and includes both local and interrepeat interactions. Our results suggest that the N-terminal capping motif serves to polarize the folding pathway by acting as a fast-growing nucleus onto which consecutive repeats fold in the transition state ensemble, and highlight the importance of sequence-specific interactions in pathway selection.
Improved Structures of Full-Length p97, an AAA ATPase: Implications for Mechanisms of Nucleotide-Dependent Conformational Change
by Jason M. Davies; Axel T. Brunger; William I. Weis (pp. 715-726).
The ATPases associated with various cellular activities (AAA) protein p97 has been implicated in a variety of cellular processes, including endoplasmic reticulum-associated degradation and homotypic membrane fusion. p97 belongs to a subgroup of AAA proteins that contains two nucleotide binding domains, D1 and D2. We determined the crystal structure of D2 at 3.0 Å resolution. This model enabled rerefinement of full-length p97 in different nucleotide states against previously reported low-resolution diffraction data to significantly improved R values and Ramachandran statistics. Although the overall fold remained similar, there are significant improvements, especially around the D2 nucleotide binding site. The rerefinement illustrates the importance of knowledge of high-resolution structures of fragments covering most of the whole molecule. The structures suggest that nucleotide hydrolysis is transformed into larger conformational changes by pushing of one D2 domain by its neighbor in the hexamer, and transmission of nucleotide-state information through the D1-D2 linker to displace the N-terminal, effector binding domain.
Structure of the Staphylococcus aureus AgrA LytTR Domain Bound to DNA Reveals a Beta Fold with an Unusual Mode of Binding
by David J. Sidote; Christopher M. Barbieri; Ti Wu; Ann M. Stock (pp. 727-735).
The LytTR domain is a DNA-binding motif found within the AlgR/AgrA/LytR family of transcription factors that regulate virulence factor and toxin gene expression in pathogenic bacteria. This previously uncharacterized domain lacks sequence similarity with proteins of known structure. The crystal structure of the DNA-binding domain of Staphylococcus aureus AgrA complexed with a DNA pentadecamer duplex has been determined at 1.6 Å resolution. The structure establishes a 10-stranded β fold for the LytTR domain and reveals its mode of interaction with DNA. Residues within loop regions of AgrA contact two successive major grooves and the intervening minor groove on one face of the oligonucleotide duplex, inducing a substantial bend in the DNA. Loss of DNA binding upon substitution of key interacting residues in AgrA supports the observed binding mode. This mode of protein-DNA interaction provides a potential target for future antimicrobial drug design.
Keywords: MICROBIO; PROTEINS; DNA
A Coupled Equilibrium Shift Mechanism in Calmodulin-Mediated Signal Transduction
by Jörg Gsponer; John Christodoulou; Andrea Cavalli; Jennifer M. Bui; Barbara Richter; Christopher M. Dobson; Michele Vendruscolo (pp. 736-746).
We used nuclear magnetic resonance data to determine ensembles of conformations representing the structure and dynamics of calmodulin (CaM) in the calcium-bound state (Ca2+-CaM) and in the state bound to myosin light chain kinase (CaM-MLCK). These ensembles reveal that the Ca2+-CaM state includes a range of structures similar to those present when CaM is bound to MLCK. Detailed analysis of the ensembles demonstrates that correlated motions within the Ca2+-CaM state direct the structural fluctuations toward complex-like substates. This phenomenon enables initial ligation of MLCK at the C-terminal domain of CaM and induces a population shift among the substates accessible to the N-terminal domain, thus giving rise to the cooperativity associated with binding. Based on these results and the combination of modern free energy landscape theory with classical allostery models, we suggest that a coupled equilibrium shift mechanism controls the efficient binding of CaM to a wide range of ligands.
Keywords: PROTEINS; SIGNALING
The Molecular Mechanism of Toxin-Induced Conformational Changes in a Potassium Channel: Relation to C-Type Inactivation
by Ulrich Zachariae; Robert Schneider; Phanindra Velisetty; Adam Lange; Daniel Seeliger; Sören J. Wacker; Yasmin Karimi-Nejad; Gert Vriend; Stefan Becker; Olaf Pongs; Marc Baldus; Bert L. de Groot (pp. 747-754).
Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.
Keywords: SIGNALING; CELLBIO
Multiple Conformations of E. coli Hsp90 in Solution: Insights into the Conformational Dynamics of Hsp90
by Kristin A. Krukenberg; Friedrich Förster; Luke M. Rice; Andrej Sali; David A. Agard (pp. 755-765).
Hsp90, an essential eukaryotic chaperone, depends upon its intrinsic ATPase activity for function. Crystal structures of the bacterial Hsp90 homolog, HtpG, and the yeast Hsp90 reveal large domain rearrangements between the nucleotide-free and the nucleotide-bound forms. We used small-angle X-ray scattering and recently developed molecular modeling methods to characterize the solution structure of HtpG and demonstrate how it differs from known Hsp90 conformations. In addition to this HtpG conformation, we demonstrate that under physiologically relevant conditions, multiple conformations coexist in equilibrium. In solution, nucleotide-free HtpG adopts a more extended conformation than observed in the crystal, and upon the addition of AMPPNP, HtpG is in equilibrium between this open state and a closed state that is in good agreement with the yeast AMPPNP crystal structure. These studies provide a unique view of Hsp90 conformational dynamics and provide a model for the role of nucleotide in effecting conformational change.
An Intersubunit Active Site between Supercoiled Parallel β Helices in the Trimeric Tailspike Endorhamnosidase of Shigella flexneri Phage Sf6
by Jürgen J. Müller; Stefanie Barbirz; Karolin Heinle; Alexander Freiberg; Robert Seckler; Udo Heinemann (pp. 766-775).
Sf6 belongs to the Podoviridae family of temperate bacteriophages that infect gram-negative bacteria by insertion of their double-stranded DNA. They attach to their hosts specifically via their tailspike proteins. The 1.25 Å crystal structure of Shigella phage Sf6 tailspike protein (Sf6 TSP) reveals a conserved architecture with a central, right-handed β helix. In the trimer of Sf6 TSP, the parallel β helices form a left-handed, coiled−β coil with a pitch of 340 Å. The C-terminal domain consists of a β sandwich reminiscent of viral capsid proteins. Further crystallographic and biochemical analyses show a Shigella cell wall O-antigen fragment to bind to an endorhamnosidase active site located between two β-helix subunits each anchoring one catalytic carboxylate. The functionally and structurally related bacteriophage, P22 TSP, lacks sequence identity with Sf6 TSP and has its active sites on single subunits. Sf6 TSP may serve as an example for the evolution of different host specificities on a similar general architecture.
Keywords: PROTEINS; MICROBIO
Partitivirus Structure Reveals a 120-Subunit, Helix-Rich Capsid with Distinctive Surface Arches Formed by Quasisymmetric Coat-Protein Dimers
by Wendy F. Ochoa; Wendy M. Havens; Robert S. Sinkovits; Max L. Nibert; Said A. Ghabrial; Timothy S. Baker (pp. 776-786).
Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 Å resolution. The capsid, ∼350 Å in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of α helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called “T = 2” capsids of other dsRNA viruses.
Keywords: MICROBIO; PROTEINS
Transmembrane Helix Uniformity Examined by Spectral Mapping of Torsion Angles
by Richard C. Page; Sanguk Kim; Timothy A. Cross (pp. 787-797).
The environment and unique balance of molecular forces within lipid bilayers has a profound impact upon the structure, dynamics, and function of membrane proteins. We describe the biophysical foundations for the remarkable uniformity of many transmembrane helices that result from the molecular interactions within lipid bilayers. In fact, the characteristic uniformity of transmembrane helices leads to unique spectroscopic opportunities allowing for φ,ψ torsion angles to be mapped directly onto solid state nuclear magnetic resonance (NMR) PISEMA spectra. Results from spectral simulations, the solid state NMR-derived structure of the influenza A M2 proton channel transmembrane domain, and high-resolution crystal structures of 27 integral membrane proteins demonstrate that transmembrane helices tend to be more uniform than previously thought. The results are discussed through the definition of a preferred range of backbone ϕ,ψ torsion angles for transmembrane α helices and are presented with respect to improving biophysical characterizations of integral membrane proteins.
Structure of a NEMO/IKK-Associating Domain Reveals Architecture of the Interaction Site
by Mia Rushe; Laura Silvian; Sarah Bixler; Ling Ling Chen; Anne Cheung; Scott Bowes; Hernan Cuervo; Steven Berkowitz; Timothy Zheng; Kevin Guckian; Maria Pellegrini; Alexey Lugovskoy (pp. 798-808).
The phosphorylation of IκB by the IKK complex targets it for degradation and releases NF-κB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKα/β kinases and a regulatory protein, NF-κB essential modulator (NEMO; IKKγ). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complex's catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.
Keywords: PROTEINS; SIGNALING
Crystal Structures of Human Saposins C and D: Implications for Lipid Recognition and Membrane Interactions
by Maxim Rossmann; Robert Schultz-Heienbrok; Joachim Behlke; Natascha Remmel; Claudia Alings; Konrad Sandhoff; Wolfram Saenger; Timm Maier (pp. 809-817).
Human saposins are essential proteins required for degradation of sphingolipids and lipid antigen presentation. Despite the conserved structural organization of saposins, their distinct modes of interaction with biological membranes are not fully understood. We describe two crystal structures of human saposin C in an “open” configuration with unusual domain swapped homodimers. This form of SapC dimer supports the “clip-on” model for SapC-induced vesicle fusion. In addition, we present the crystal structure of SapD in two crystal forms. They reveal the monomer-monomer interface for the SapD dimer, which was confirmed in solution by analytical ultracentrifugation. The crystal structure of SapD suggests that side chains of Lys10 and Arg17 are involved in initial association with the preferred anionic biological membranes by forming salt bridges with sulfate or phosphate lipid headgroups.
Keywords: PROTEINS; CELLBIO
Structural Plasticity Underpins Promiscuous Binding of the Prosurvival Protein A1
by Callum Smits; Peter E. Czabotar; Mark G. Hinds; Catherine L. Day (pp. 818-829).
Apoptotic pathways are regulated by protein-protein interactions. Interaction of the BH3 domains of proapoptotic Bcl-2 family proteins with the hydrophobic groove of prosurvival proteins is critical. Whereas some BH3 domains bind in a promiscuous manner, others exhibit considerable selectivity and the sequence characteristics that distinguish these activities are unclear. In this study, crystal structures of complexes between the prosurvival protein A1 and the BH3 domains from Puma, Bmf, Bak, and Bid have been solved. The structure of A1 is similar to that of other prosurvival proteins, although features, such as an acidic patch in the binding groove, may allow specific therapeutic modulation of apoptosis. Significant conformational plasticity was observed in the intermolecular interactions and these differences explain some of the variation in affinity. This study, in combination with published data, suggests that interactions between conserved residues demarcate optimal binding.