Structure (v.20, #4)

In This Issue (v-vii).

Human Tripeptidyl Peptidase II: A Gentle Giant by Robert M. Glaeser (565-566).
Molecular structures can serve to either validate or rule out existing hypotheses, and they can also spawn new, deeper proposals about biochemical mechanism. In this issue of Structure, Schönegge et al. use single-particle cryo-electron microscopy and flexible docking to examine the function of human tripeptidyl peptidase II, including the role of conformational changes in enzyme activation.

XPF-ERCC1: On the Bubble by Steven M. Shell; Walter J. Chazin (566-568).
In this issue of Structure, Das et al. report the structure of the helix-hairpin-helix dimerization domain of XPF bound to ssDNA. These results provide insight into the architecture of nucleotide excision repair machinery and how it interacts with damaged DNA substrates.

Discoidin Discoveries by Kathryn M. Ferguson (568-570).
In this issue of Structure, Carafoli et al. investigate the mode of antibody-mediated inhibition of the discoidin domain receptor 1 (DDR1). These studies also provide new insight into activation of the DDRs, which are unique among receptor tyrosine kinases in the composition of their extracellular regions.

Phosphorylation Meets Proteolysis by Martin Renatus; Christopher J. Farady (570-571).
Phosphorylation is a reversible post-translational modification that regulates many proteins and enzymes, including proteases, as shown by two recent publications. Huang and colleagues and Velázquez-Delgado and Hardy (this issue of Structure) describe how phosphorylation activates the protease activity of the deubiquitinating enzyme DUBA and how it inhibits caspase-6, respectively.

Methionine Scanning as an NMR Tool for Detecting and Analyzing Biomolecular Interaction Surfaces by Mira C. Stoffregen; Matthias M. Schwer; Fabian A. Renschler; Silke Wiesner (573-581).
Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.

Fabs Enable Single Particle cryoEM Studies of Small Proteins by Shenping Wu; Agustin Avila-Sakar; JungMin Kim; David S. Booth; Charles H. Greenberg; Andrea Rossi; Maofu Liao; Xueming Li; Akram Alian; Sarah L. Griner; Narinobu Juge; Yadong Yu; Claudia M. Mergel; Javier Chaparro-Riggers; Pavel Strop; Robert Tampé; Robert H. Edwards; Robert M. Stroud; Charles S. Craik; Yifan Cheng (582-592).
In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an ∼65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.► An approach of enabling single particle cryoEM of small proteins by Fabs ► A rigidly bound Fab is a fiducial marker to facilitate accurate image alignment ► A well-resolved Fab density is an internal control of the 3D reconstruction ► A 3D reconstruction of a 65 kDa protein in complex with Fabs by single particle cryoEM

The Structure of Human Tripeptidyl Peptidase II as Determined by a Hybrid Approach by Anne-Marie Schönegge; Elizabeth Villa; Friedrich Förster; Reiner Hegerl; Jürgen Peters; Wolfgang Baumeister; Beate Rockel (593-603).
Tripeptidyl-peptidase II (TPPII) is a high molecular mass (∼5 MDa) serine protease, which is thought to act downstream of the 26S proteasome, cleaving peptides released by the latter. Here, the structure of human TPPII (HsTPPII) has been determined to subnanometer resolution by cryoelectron microscopy and single-particle analysis. The complex is built from two strands forming a quasihelical structure harboring a complex system of inner cavities. HsTPPII particles exhibit some polymorphism resulting in complexes consisting of nine or of eight dimers per strand. To obtain deeper insights into the architecture and function of HsTPPII, we have created a pseudoatomic structure of the HsTPPII spindle using a comparative model of HsTPPII dimers and molecular dynamics flexible fitting. Analyses of the resulting hybrid structure of the HsTPPII holocomplex provide new insights into the mechanism of maturation and activation.► Method for recombinant expression and purification of human TPPII ► 3D map of human TPPII at 9–10 Å resolution by cryo-EM ► Pseudoatomic model of human TPPII by flexible fitting of comparative models ► Dimer-dimer contacts trigger changes in the active site and the entrance to the cavity system

Solution Structure Analysis of the HPV16 E6 Oncoprotein Reveals a Self-Association Mechanism Required for E6-Mediated Degradation of p53 by Katia Zanier; Abdellahi ould M'hamed ould Sidi; Charlotte Boulade-Ladame; Vladimir Rybin; Anne Chappelle; Andrew Atkinson; Bruno Kieffer; Gilles Travé (604-617).
The viral oncoprotein E6 is an essential factor for cervical cancers induced by “high-risk” mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.Display Omitted► E6 self-association occurs via dimerization of its N-terminal (E6N) domain ► Structures of the monomeric E6 domains and of the E6N homodimer are presented ► Mutations at the E6N dimer interface inactivate E6 for p53 degradation activity ► A correlation between E6 self-association and p53 degradation activities is observed

How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations by Serdal Kirmizialtin; Virginia Nguyen; Kenneth A. Johnson; Ron Elber (618-627).
Nearly every enzyme undergoes a significant change in structure after binding it's substrate. Experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a millisecond timescale locks the correct nucleotide at the active site but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, whereas the same steps diminish the reaction probability of an incorrect substrate.► Kinetics and atomic simulations quantitatively explain nucleotide binding to HIV-RT ► Reaction path, free energy, and kinetics of HIV-RT transition are computed ► Simulations show that specificity is based on induced fit ► Specificity is determined by the rate of conformation change not only chemical step

Dissecting the Kinematics of the Kinesin Step by Zhechun Zhang; D. Thirumalai (628-640).
Kinesin walks processively on microtubules in an asymmetric hand-over-hand manner with each step spanning 16 nm. We used molecular simulations to determine the fraction of a single step due to conformational changes in the neck linker, and that due to diffusion of the tethered head. Stepping is determined largely by two energy scales, one favoring neck-linker docking and the other, εh MT-TH, between the trailing head (TH) and the microtubule. Neck-linker docking and an optimal value of εh MT-TH are needed to minimize the probability that the TH takes side steps. There are three major stages in the kinematics of a step. In the first, the neck linker docks, resulting in ∼(5–6) nm movements of the trailing head. The TH moves an additional (6–8) nm in stage II by anisotropic translational diffusion. In the third stage, spanning ∼(3–4) nm, the step is complete with the TH binding to the αβ-tubulin binding site.Display Omitted► Neck-linker docking moves the trailing head by about 6 nm ► Optimal MT-kinesin interaction and neck-linker docking minimize side step probability ► Search for the target binding site involves stochastic search ► Jump dynamics and search for the target binding site occur in three major stages

The interconversion of actin between monomeric and polymeric forms is a fundamental process in cell biology that is incompletely understood, in part because there is no high-resolution structure for filamentous actin. Several models have been proposed recently; identifying structural and dynamic differences between them is an essential step toward understanding actin dynamics. We compare three of these models, using coarse-grained analysis of molecular dynamics simulations to analyze the differences between them and evaluate their relative stability. Based on this analysis, we identify key motions that may be associated with polymerization, including a potential energetic barrier in the process. We also find that actin subunits are polymorphic; during simulations they assume a range of configurations remarkably similar to those seen in recent cryoEM images.Display Omitted► Coarse-grained analysis of MD simulations is used to compare F-actin models ► After simulation, most subunits resemble the Oda model; strands are closer together ► Subunit flattening is coupled to a hinging motion of subdomain 2 ► After equilibration, F-actin models differ mainly in the position of subdomain 2

Asymmetric Mode of Ca2+-S100A4 Interaction with Nonmuscle Myosin IIA Generates Nanomolar Affinity Required for Filament Remodeling by Paul R. Elliott; Andrew F. Irvine; Hyun Suk Jung; Kaeko Tozawa; Martyna W. Pastok; Remigio Picone; Sandip K. Badyal; Jaswir Basran; Philip S. Rudland; Roger Barraclough; Lu-Yun Lian; Clive R. Bagshaw; Marina Kriajevska; Igor L. Barsukov (654-666).
Filament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca2+-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca2+-dependent binding sites of each subunit in the dimer. Synergy between these sites leads to a very tight association (KD ∼1 nM) that is unique in the S100 family. Single-residue mutations that remove this synergy weaken binding and ameliorate the effects of S100A4 on NMIIA filament assembly and cell spreading in A431 human epithelial carcinoma cells. We propose a model for NMIIA filament disassembly by S100A4 in which initial binding to the unstructured NMIIA tail initiates unzipping of the coiled coil and disruption of filament packing.Display Omitted► S100A4 dimer binds single-myosin IIA molecule in an asymmetrical mode ► Synergy between Ca-dependent sites in S100A4 leads to high-affinity interaction ► Electron microscopy shows that two S100A4 dimers bind to the myosin coiled coil ► S100A4 has direct effect on formation of stress fibers and cell migration

The Structure of the XPF-ssDNA Complex Underscores the Distinct Roles of the XPF and ERCC1 Helix- Hairpin-Helix Domains in ss/ds DNA Recognition by Devashish Das; Gert E. Folkers; Marc van Dijk; Nicolaas G.J. Jaspers; Jan H.J. Hoeijmakers; Robert Kaptein; Rolf Boelens (667-675).
Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5′ end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction.Display Omitted► The C-terminal domain of XPF binds preferentially ssDNA ► The complex of XPF and ssDNA has a well-defined structure ► The XPF-ssDNA interaction contains a direct guanine base contact ► Together, ERCC1 and XPF position XPF/ERCC1 at the ss/ds DNA junction

Structural Basis for the Dual Recognition of Helical Cytokines IL-34 and CSF-1 by CSF-1R by Xiaolei Ma; Wei Yu Lin; Yongmei Chen; Scott Stawicki; Kiran Mukhyala; Yan Wu; Flavius Martin; J. Fernando Bazan; Melissa A. Starovasnik (676-687).
Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.► Structure of human IL-34 reveals a dimeric four-helix bundle cytokine fold ► Studies in solution confirm bivalent assembly and high affinity of IL-34 with CSF-1R ► The IL-34/CSF-1R complex structure shares a common architecture with CSF-1/CSF-1R ► CSF-1R employs distinct domain adaptations for binding to IL-34 and CSF-1

Structure of the Discoidin Domain Receptor 1 Extracellular Region Bound to an Inhibitory Fab Fragment Reveals Features Important for Signaling by Federico Carafoli; Marie Cathrin Mayer; Kazushige Shiraishi; Mira Anguelova Pecheva; Lai Yi Chan; Ruodan Nan; Birgit Leitinger; Erhard Hohenester (688-697).
The discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding. The crystal structure of the monomeric DDR1 extracellular region bound to the Fab fragment of mAb 3E3 reveals that the collagen-binding discoidin (DS) domain is tightly associated with the following DS-like domain, which contains the epitopes of all mAbs. A conserved surface patch in the DS domain outside the collagen-binding site is shown to be required for signaling. Thus, the active conformation of the DDR1 dimer involves collagen-induced contacts between the DS domains, in addition to the previously identified association of transmembrane helices. The mAbs likely inhibit signaling by sterically blocking the extracellular association of DDR1 subunits.Display Omitted► Monoclonal antibodies inhibit DDR1 signaling without blocking collagen binding ► The DDR1 extracellular region consists of a DS and a DS-like domain ► The collagen-binding DS domain contains a patch that is essential for signaling ► The mAbs bind to the DS-like domain, preventing formation of the active DDR dimer

Crystal Structures of Aureochrome1 LOV Suggest New Design Strategies for Optogenetics by Devrani Mitra; Xiaojing Yang; Keith Moffat (698-706).
Aureochrome1, a signaling photoreceptor from a eukaryotic photosynthetic stramenopile, confers blue-light-regulated DNA binding on the organism. Its topology, in which a C-terminal LOV sensor domain is linked to an N-terminal DNA-binding bZIP effector domain, contrasts with the reverse sensor-effector topology in most other known LOV-photoreceptors. How, then, is signal transmitted in Aureochrome1? The dark- and light-state crystal structures of Aureochrome1 LOV domain (AuLOV) show that its helical N- and C-terminal flanking regions are packed against the external surface of the core β sheet, opposite to the FMN chromophore on the internal surface. Light-induced conformational changes occur in the quaternary structure of the AuLOV dimer and in Phe298 of the Hβ strand in the core. The properties of AuLOV extend the applicability of LOV domains as versatile design modules that permit fusion to effector domains via either the N- or C-termini to confer blue-light sensitivity.Display Omitted► Crystal structures in dark and light states determined for Aureochrome1 LOV ► Significant light induced change observed in F298 and quaternary structure ► Potential photocycle mutant variants, altering the light-state lifetime, identified ► Possible mechanism of signaling in topologically unique Aureo1 speculated

Mucin glycoproteins with large numbers of O-linked glycosylations comprise the mucosal barrier lining the mammalian gastrointestinal tract from mouth to gut. A critical biological function of mucins is to protect the underlying epithelium from infection. Enterohemorrhagic Escherichia coli (EHEC), the mediator of severe food- and water-borne disease, can breach this barrier and adhere to intestinal cells. StcE, a ∼100 kDa metalloprotease secreted by EHEC, plays a pivotal role in remodeling the mucosal lining during infection. To obtain mechanistic insight into its function, we have determined the structure of StcE. Our data reveal a dynamic, multidomain architecture featuring an unusually large substrate-binding cleft and a prominent polarized surface charge distribution highly suggestive of an electrostatic role in substrate targeting. The observation of key conserved motifs in the active site allows us to propose the structural basis for the specific recognition of α-O-glycan-containing substrates. Complementary biochemical analysis provides further insight into its distinct substrate specificity and binding stoichiometry.► StcE is a multidomain O-glycoprotein-specific metalloprotease ► Unique active site features allow for accommodation of highly glycosylated substrates ► 1:1 stoichiometry of binding to the host target C1-esterase inhibitor ► StcE has a subsite preference for acidic residues at the P1 position

Detection of Spatial Correlations in Protein Structures and Molecular Complexes by Manfred J. Sippl; Markus Wiederstein (718-728).
Protein structures are frequently related by spectacular and often surprising similarities. Structural correlations among protein chains are routinely detected by various structure-matching techniques, but the comparison of oligomers and molecular complexes is largely uncharted territory. Here we solve the structure-matching problem for oligomers and large molecular aggregates, including the largest molecular complexes known today. We provide several challenging examples that cannot be handled by conventional structure-matching techniques and we report on a number of remarkable correlations. The examples cover the cell-puncturing device of bacteriophage T4, the secretion system of P. aeruginosa, members of the dehydrogenase family, DNA clamps, ferredoxin iron-storage cages, and virus capsids.► Structure-matching techniques reveal striking similarities among protein complexes ► Large-scale structural transitions in the evolution of the dehydrogenase family ► Similar molecular shapes built from distinct molecular components ► Exceptional structural correlations among virus capsids

The osmoregulator trimethylamine-N-oxide (TMAO), commonplace in aquatic organisms, is used as the terminal electron acceptor for respiration in many bacterial species. The TMAO reductase (Tor) pathway for respiratory catalysis is controlled by a receptor system that comprises the TMAO-binding protein TorT, the sensor histidine kinase TorS, and the response regulator TorR. Here we study the TorS/TorT sensor system to gain mechanistic insight into signaling by histidine kinase receptors. We determined crystal structures for complexes of TorS sensor domains with apo TorT and with TorT (TMAO); we characterized TorS sensor associations with TorT in solution; we analyzed the thermodynamics of TMAO binding to TorT-TorS complexes; and we analyzed in vivo responses to TMAO through the TorT/TorS/TorR system to test structure-inspired hypotheses. TorS-TorT(apo) is an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase.Display Omitted► ToT-TorS sensor complex structures are presented in both apo and ligated states ► The ToT-TorS sensor system acts with an atypical form of negative cooperativity ► Signal transduction appears to involve an asymmetry-to-symmetry switch

Phosphorylation Regulates Assembly of the Caspase-6 Substrate-Binding Groove by Elih M. Velázquez-Delgado; Jeanne A. Hardy (742-751).
Caspases, a family of apoptotic proteases, are increasingly recognized as being extensively phosphorylated, usually leading to inactivation. To date, no structural mechanism for phosphorylation-based caspase inactivation is available, although this information may be key to achieving caspase-specific inhibition. Caspase-6 has recently been implicated in neurodegenerative conditions including Huntington's and Alzheimer's diseases. A full understanding of caspase-6 regulation is crucial to caspase-6-specific inhibition. Caspase-6 is phosphorylated by ARK5 kinase at serine 257 leading to suppression of cell death via caspase-6 inhibition. Our structure of the fully inactive phosphomimetic S257D reveals that phosphorylation results in a steric clash with P201 in the L2′ loop. Removal of the proline side chain alleviates the clash resulting in nearly wild-type activity levels. This phosphomimetic-mediated steric clash causes misalignment of the substrate-binding groove, preventing substrate binding. Substrate-binding loop misalignment appears to be a widely used regulatory strategy among caspases and may present a new paradigm for caspase-specific control.Display Omitted► Phosphorylation at S257 inhibits both active caspase-6 and the procaspase-6 zymogen ► Steric clash between phosphoserine 257 and proline 201 inhibits caspase-6 ► Steric clash causes misalignment of the four substrate-binding groove loops ► Loop misalignment by phosphorylation is predicted for many caspases