2-Cys peroxiredoxins (Prxs) play two different roles depending on the physiological status of the cell. They are thioredoxin-dependent peroxidases under low oxidative stress, and ATP-independent chaperones upon exposure to high peroxides concentrations. These alternative functions have been associated with changes in the oligomerization state from low (LMW) to high (HMW) molecular weight species. Here we present the structures of Schistosoma mansoni PrxI in both states: the LMW decamer and the HMW 20-mer, formed by two stacked decamers. The latter is the first structure of a 2-Cys Prx chaperonic form. Comparison of the structures sheds light on the mechanism by which chemical stressors, such as high H2O2 concentration and acidic pH, are sensed and translated into a functional switch in this protein family. We also propose a model to account for the in vivo formation of long filaments of stacked Prx rings.
The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here we report single particle cryo-EM-derived structure of the supra-molecular assembly comprising of S. cerevisiae ORC, the replication initiation factor Cdc6 and double strand ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3 with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, particularly re-orientating the Orc1 N-terminal BAH-domain. Segmentation of the 3D map of ORC•Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.
We report the sub nanometer cryo-electron microscopy (cryoEM) reconstruction of a novel marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species with significant ecological importance including the broadly antagonistic bacterium Vibrio sp. SWAT3. The three dimensional structure of the 800Å SIO-2, icosahedrally averaged, head of the tailed particle revealed a novel T=12 quasi-symmetry not previously described in a bacteriophage. Two, morphologically distinct, types of auxiliary proteins were also identified; one species bound to the surface of hexamers and the other bound to pentamers. The secondary structure, evident in the electron density, shows that the major capsid protein has the HK97-like fold. The three-dimensional structure of the procapsid form, also presented here, has no “decoration” proteins and reveals a novel capsomer organization due to the constraints of the T=12 symmetry.
Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4 Å and 10 Å, either by cryo-electron microscopy (cryoEM) or X-ray crystallography. In these density maps the general arrangement of secondary structure elements is revealed while their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the secondary structure elements de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins the protocol consistently and reproducibly achieves models with RMSD values smaller than 3 Å.
A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5–6Å, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses a novel approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallography, pathwalking can become an important tool in modeling protein structures.
The ESCRTs play multiple roles within the cell, including degradation of ubiquitinated membrane proteins by the sorting them into multivesicular bodies (MVBs). Two recent studies provide structural and functional insights into how the newly identified ESCRT-I component UBAP1 dedicates ESCRT-I function for sorting ubiquitinated proteins at the MVB (Agromayor et al., 2012; Stefani et al. 2011).
Variable Lymphocyte Receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin-fold based T- and B-cell receptors, lymphocyte-like cells of jawless fish express VLRs (A, B or C) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA, C) in function. Here we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.
Rhomboid proteases regulate key cellular pathways, but their biochemical mechanism including how water is made available to the membrane-immersed active site remains ambiguous. We performed four prolonged molecular dynamics simulations initiated from both gate-open and gate-closed states of Escherichia coli rhomboid GlpG in a phospholipid bilayer. GlpG was notably stable in both gating states, experiencing similar tilt and local membrane thinning, with no observable gating transitions, highlighting that gating is rate-limiting. Analysis of dynamics revealed rapid loss of crystallographic waters from the active site, but retention of a water cluster within a site formed by His141, Ser181, Ser185 and/or Gln189. Experimental interrogation of 14 engineered mutants revealed an essential role for at least Gln189 and Ser185 in catalysis with no effect on structural stability. Our studies indicate that spontaneous water supply to the intra-membrane active site of rhomboid proteases is rare, but its availability is ensured by an unanticipated active site element, the water-retention site.
rhomboid protease; molecular dynamics simulation; lipid bilayer; water retention site; transmembrane helix; intramembrane proteolysis
While disordered to ordered rearrangements are relatively common, the ability of proteins to switch from one ordered fold to a completely different fold is generally regarded as rare and few fold switches have been characterized. Here, in a designed system, we examine the mutational requirements for transitioning between folds and functions. We show that switching between monomeric 3α and 4β+α folds can occur in multiple ways with successive single amino acid changes at diverse residue positions, raising the likelihood that such transitions occur in the evolution of new folds. Even mutations on the periphery of the core can tip the balance between alternatively folded states. Ligand-binding studies illustrate that a new IgG-binding function can be gained well before the relevant 4β+α fold is appreciably populated in the unbound protein. The results provide new insights into the evolution of fold and function.
An exposed F-type lectin domain fused to the N-terminus of a cholesterol-dependent cytolysin scaffold allows Streptococcus mitis Lectinolysin to cluster at fucose-rich sites on target cell membranes, thereby leading to increased pore-forming toxin activity. In this issue of Structure, Feil and coworkers define the structural basis for Lectinolysin glycan-binding specificity (Feil et al, 2012).
The Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The "RAM" region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly of a MAML1/NICD/CSL complex using HX-MS. Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2–3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired site DNA.
Identification of homogeneous subsets of images in a macromolecular electron microscopy (EM) image data set is a critical step in single-particle analysis. The task is handled by iterative algorithms, whose performance is compromised by the compounded limitations of image alignment and K-means clustering. Here we describe an approach, iterative stable alignment and clustering (ISAC) that, relying on a new clustering method and on the concepts of stability and reproducibility, can extract validated, homogeneous subsets of images. ISAC requires only a small number of simple parameters and, with minimal human intervention, can eliminate bias from two-dimensional image clustering and maximize the quality of group averages that can be used for ab initio three-dimensional structural determination and analysis of macromolecular conformational variability. Repeated testing of the stability and reproducibility of a solution within ISAC eliminates heterogeneous or incorrect classes and introduces critical validation to the process of EM image clustering.
Successful targets for anti-cancer drugs such as clofarabine and gemcitabine, ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNR as well as present a mechanism for the formation of these unusual structures.
The GET pathway, using several proteins (Gets 1–5 and probably Sgt2), posttranslationally conducts tail-anchored (TA) proteins to the endoplasmic reticulum (ER). At the ER, TA proteins are inserted into the lipid bilayer and then sorted and directed to their respective destinations in the secretory pathway. Until last year, there was no structural information on any of the GET components but now there are ten crystal structures of Get3 in a variety of nucleotide-bound states and conformations. The structures of Get4 and a portion of Get5 also emerged in 2010. This minireview provides a detailed comparison of the GET structures and discusses their mechanistic relevance to TA protein insertion. It also addresses the outstanding gaps in detailed molecular information on this system, including the structures of Get5, Sgt2, and the transmembrane complex comprising Get1 and Get2.
The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that mediates cotranslational targeting of secreted and membrane proteins to the membrane. Targeting is regulated by GTP binding and hydrolysis events that require direct interaction between structurally homologous “NG” GTPase domains of the SRP signal recognition subunit and its membrane-associated receptor, SRα. Structures of both the apo and GDP bound NG domains of the prokaryotic SRP54 homolog, Ffh, and the prokaryotic receptor homolog, FtsY, have been determined. The structural basis for the GTP-dependent interaction between the two proteins, however, remains unknown.
We report here two structures of the NG GTPase of Ffh from Thermus aquaticus bound to the nonhydrolyzable GTP analog GMPPNP. Both structures reveal an unexpected binding mode in which the β-phosphate is kinked away from the binding site and magnesium is not bound. Binding of the GTP analog in the canonical conformation found in other GTPase structures is precluded by constriction of the phosphate binding P loop. The structural difference between the Ffh complex and other GTPases suggests a specific conformational change that must accompany movement of the nucleotide from an “inactive” to an “active” binding mode.
Conserved side chains of the GTPase sequence motifs unique to the SRP subfamily may function to gate formation of the active GTP bound conformation. Exposed hydrophobic residues provide an interaction surface that may allow regulation of the GTP binding conformation, and thus activation of the GTPase, during the association of SRP with its receptor.
SRP; Ffh; NG domain; GTPase; GMPPNP; X-ray crystallography
MRG15 is a member of the mortality family of transcription factors that targets a wide variety of multi-protein complexes involved in transcription regulation, DNA repair, and alternative splicing to chromatin. The structure of the apo-MRG15 MRG domain implicated in interactions with diverse proteins has been described, but not in complex with any of its targets. Here we structurally and functionally characterize the interaction between MRG15 and Pf1, two constitutively-associated subunits of the histone deacetylase-associated Rpd3S/Sin3S corepressor complex. The MRG domain adopts a structure reminiscent of the apo-state whereas the Pf1 MRG-binding domain engages two discrete hydrophobic surfaces on the MRG domain via a bipartite motif comprising an α-helix and a segment in an extended conformation, both of which are critical for high-affinity interactions. Multiple MRG15 interactors share an FxLP motif in the extended segment but equivalent sequence/helical motifs are not readily evident, implying potential diversity in MRG-recognition mechanisms.
We demonstrate how Tryptophanyl-tRNA synthetase (TrpRS) uses conformation-dependent Mg2+ activation to couple catalysis of tryptophan activation to specific, functional domain movements. Rate acceleration by Mg2+ requires ~ -6.0 kcal/mole in protein•Mg2+ interaction energy, none of which arises from the active site. A highly cooperative interaction between Mg2+ and four residues from a remote, conserved motif that mediates the shear of domain movement: (i) destabilizes the pre-transition state conformation, thereby (ii) inducing the Mg2+ to stabilize the transition state for kcat by ~ -5.0 kcal/mole. Cooperative, long-range conformational effects on the metal therefore convert an inactive Mg2+ coordination into one that can stabilize the transition state if, and only if, domain motion occurs. Transient, conformation-dependent Mg2+ activation, analogous to the escapement in mechanical clocks, explains vectorial coupling.
Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally-based test cases: Tobacco mosaic virus, a rod-shaped virus; and aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multi-component structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.
AFM; protein structure; integrative modeling; molecular assembly; super-complexes; computational biology
Regulated relocalization of signaling and trafficking proteins is crucial for the control of many cellular processes, and is driven by a series of domains that respond to alterations at membrane surfaces. The first examples of these domains – conditional peripheral membrane proteins – included C1, C2, PH, PX, and FYVE domains, which specifically recognize single tightly regulated membrane components such as diacylglycerol or phosphoinositides. The structural basis for this recognition is now well understood. Efforts to identify additional domains with similar functions that bind other targets (or participate in unexplained cellular processes) have not yielded many more examples of specific phospholipid-binding domains. Instead, most of the recently discovered conditional peripheral membrane proteins bind multiple targets (each with limited specificity), relying on coincidence detection, and/or recognizing broader physical properties of the membrane such as charge or curvature. This broader range of recognition modes presents significant methodological challenges for a full structural understanding.
Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution NMR studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators.
The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural bases for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and from other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex towards alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM whereas EphB members may not; correspondingly we demonstrate binding of EphA1 but not of EphB2 to SHIP2 SAM. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting chemotactic cell migration in coordination with the receptor.
NMR structure determination of a protein complex; molecular dynamics and docking calculations; binding thermodynamics and specificity; receptor tyrosine kinase; SHIP2; cell migration; endocytosis
The newly discovered FabV enoyl-ACP reductase, which catalyzes the last step of the bacterial fatty acid biosynthesis (FAS-II) pathway, is a promising but unexploited drug target against the re-emerging pathogen Yersinia pestis. The structure of the Y. pestis FabV in complex with its cofactor reveals that the enzyme features the common architecture of the short chain dehydrogenase reductase superfamily, but contains additional structural elements which are mostly folded around the usually flexible substrate binding loop, thereby stabilizing it in a very tight conformation that seals the active site. The structures of FabV in complex with NADH and two newly developed 2-pyridone inhibitors provide first insights for the development of new lead compounds, and suggest a mechanism by which the substrate binding-loop opens to admit the inhibitor, a motion that could also be coupled to the interaction of FabV with the acyl-carrier-protein substrate.
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human malignancies necessitating multiple layers of self-regulatory control mechanisms. Fibroblast growth factor receptor (FGFR) autoinhibition mediated by the alternatively spliced immunoglobulin (Ig) domain 1 (D1) and the acid box (AB)-containing linker between D1 and Ig domain 2 (D2) serves as the first line of defense to minimize inadvertent FGF signaling. In this report, nuclear magnetic resonance and surface plasmon resonance spectroscopy are used to demonstrate that the AB subregion of FGFR electrostatically engages the heparan sulfate (HS)-binding site on the D2 domain in cis to directly suppress HS-binding affinity of FGFR. Furthermore, the cis electrostatic interaction sterically autoinhibits ligand-binding affinity of FGFR because of the close proximity of HS-binding and primary ligand-binding sites on the D2 domain. These data, together with the strong amino acid sequence conservation of the AB subregion among FGFR orthologs, highlight the universal role of the AB subregion in FGFR autoinhibition.
Drosophila melanogaster Toll is the founding member of an important family of pathogen-recognition receptors in humans, the Toll-like receptor (TLR) family. In contrast, the prototypical receptor is a cytokine-like receptor for Spätzle (Spz) protein and plays a dual role in both development and immunity. Here, we present the crystal structure of the N-terminal domain of the receptor that encompasses the first 201 amino acids at 2.4 Å resolution. To our knowledge, the cysteine-rich cap adopts a novel fold unique to Toll-1 orthologs in insects and that is not critical for ligand binding. However, we observed that an antibody directed against the first ten LRRs blocks Spz signaling in a Drosophila cell-based assay. Supplemented by point mutagenesis and deletion analysis, our data suggests that the region up to LRR 14 is involved in Spz binding. Comparison with mammalian TLRs reconciles previous contradictory findings about the mechanism of Toll activation.
► The N-terminal cap of Toll adopts a new fold ► It resembles a duplicated beta hairpin structure with three disulphide bonds ► Toll LRRNT cap is not critical for Spätzle binding ► The first 13 LRRs are sufficient for Spätzle binding
Gangloff et al. describe a crystal structure of the N-terminal domain of the prototypical drosophila Toll receptor at 2.4 Å resolution. The cysteine-rich N-terminal cap adopts a new fold unique to Toll-1 orthologues in insects. Contrary to expectations, it is not critically involved in Späzle binding.
Protein-peptide interactions play important roles in many cellular processes, including signal transduction, trafficking, and immune recognition. Protein conformational changes upon binding, an ill-defined peptide binding surface, and the large number of peptide degrees of freedom make the prediction of protein-peptide interactions particularly challenging. To address these challenges, we perform rapid molecular dynamics simulations in order to examine the energetic and dynamic aspects of protein-peptide binding. We find that, in most cases, we recapitulate the native binding sites and native-like poses of protein-peptide complexes. Inclusion of electrostatic interactions in simulations significantly improves the prediction accuracy. Our results also highlight the importance of protein conformational flexibility, especially side-chain movement, which allows the peptide to optimize its conformation. Our findings not only demonstrate the importance of sufficient sampling of the protein and peptide conformations, but also reveal the possible effects of electrostatics and conformational flexibility on peptide recognition.