Characterization of 14-3-3 proteins from Cryptosporidium parvum

The parasite Cryptosporidium parvum has three 14-3-3 proteins: Cp14ε, Cp14a and Cp14b, with only Cp14ε similar to human 14-3-3 proteins in sequence, peptide-binding properties and structure. Structurally, Cp14a features the classical 14-3-3 dimer but with a uniquely wide pocket and a disoriented RRY triad potentially incapable of binding phosphopeptides. The Cp14b protein deviates from the norm significantly: (i) In one subunit, the phosphorylated C-terminal tail is bound in the binding groove like a phosphopeptide. This supports our binding study indicating this protein was stabilized by a peptide mimicking its last six residues. (ii) The other subunit has eight helices instead of nine, with αA and αB forming a single helix and occluding the peptide-binding cleft. (iii) The protein forms a degenerate dimer with the two binding grooves divided and facing opposite directions. These features conspire to block and disrupt the bicameral substrate-binding pocket, suggesting a possible tripartite auto-regulation mechanism that has not been observed previously. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.     

Crystal Structure of the ATPase Domain of the Human AAA+ Protein Paraplegin/SPG7

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 Å. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations.

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Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom,HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.

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Structural Comparison of Human Mammalian Ste20-Like Kinases

Background

The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains.

Principal Findings

We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family.

Significance

The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design.

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Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIδ/Ca²⁺/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIδ/Ca²⁺/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix αD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.

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Structure of a C. perfringens Enterotoxin Mutant in Complex with a Modified Claudin-2 Extracellular Loop 2

CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.     

Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations.

We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base and the associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. With one exception we find that for all globin sequences one of the known globin folds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structural research and future development of our approach.     

Browsing the SLoop database of structurally classified loops connecting elements of protein secondary structure

We describe a web server, which provides easy access to the SLoop database of loop conformations connecting elements of protein secondary structure. The loops are classified according to their length, the type of bounding secondary structures and the conformation of the mainchain. The current release of the database consists of over 8000 loops of up to 20 residues in length. A loop prediction method, which selects conformers on the basis of the sequence and the positions of the elements of secondary structure, is also implemented. These web pages are freely accessible over the internet at http://www-cryst.bioc.cam.ac.uk/ approximately sloop.     

Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE

Background

UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family.

Principal Findings

We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location – dimer interface, interlobar helices, protein surface, or within other secondary structural elements.

Conclusions

The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme.

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Structure of the retinal determination protein Dachshund reveals a DNA binding motif

The Dachshund proteins are essential components of a regulatory network controlling cell fate determination. They have been implicated in eye, limb, brain, and muscle development. These proteins cannot be assigned to any recognizable structural or functional class based on amino acid sequence analysis. The 1.65 A crystal structure of the most conserved domain of human DACHSHUND is reported here. The protein forms an alpha/beta structure containing a DNA binding motif similar to that found in the winged helix/forkhead subgroup of the helix-turn-helix family. This unexpected finding alters the previously proposed molecular models for the role of Dachshund in the eye determination pathway. Furthermore, it provides a rational framework for future mechanistic analyses of the Dachshund proteins in several developmental contexts.     

Oligomeric structure of the MALT1 tandem Ig-like domains

Background

Mucosa-associated lymphoid tissue 1 (MALT1) plays an important role in the adaptive immune program. During TCR- or BCR-induced NF-κB activation, MALT1 serves to mediate the activation of the IKK (IκB kinase) complex, which subsequently regulates the activation of NF-κB. Aggregation of MALT1 is important for E3 ligase activation and NF-κB signaling.

Principal Findings

Unlike the isolated CARD or paracaspase domains, which behave as monomers, the tandem Ig-like domains of MALT1 exists as a mixture of dimer and tetramer in solution. High-resolution structures reveals a protein-protein interface that is stabilized by a buried surface area of 1256 Ų and contains numerous hydrogen and salt bonds. In conjunction with a second interface, these interactions may represent the basis of MALT1 oligomerization.

Conclusions

The crystal structure of the tandem Ig-like domains reveals the oligomerization potential of MALT1 and a potential intermediate in the activation of the adaptive inflammatory pathway.

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Dead-End Based Modeling Tools to Explore the Sequence Space That Is Compatible with a Given Scaffold

The dead-end elimination algorithm has proven to be a powerful tool in protein homology modeling since it allows one to determine rapidly the global minimum-energy conformation (GMEC) of an arbitrarily large collection of side chains, given fixed backbone coordinates. After introducing briefly the necessary background, we focus on logic arguments that increase the efficacy of the dead-end elimination process. Second, we present new theoretical considerations on the use of the dead-end elimination method as a tool to identify sequences that are compatible with a given scaffold structure. Third, we initiate a search for properties derived from the computed GMEC structure to predict whether a given sequence can be well packed in the core of a protein. Three properties will be considered: the nonbonded energy, the accessible surface area, and the extent by which the GMEC side-chain conformations deviate from a locally optimal conformation.     

Construction and validation of a homology model of the human voltage-gated proton channel hHV1

The topological similarity of voltage-gated proton channels (H_V1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether H_V1s have a similar structure. We present the construction and validation of a homology model of the human H_V1 (hH_V1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hH_V1 by exploiting the homology of hH_V1 with VSDs of K⁺ and Na⁺ channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hH_V1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp¹¹². Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hH_V1. Each Arg residue in the S4 helix of hH_V1 was replaced by His to test accessibility using Zn²⁺ as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg²¹¹, the third Arg residue in the S4 helix in hH_V1, remains accessible to the internal solution and is located near the charge transfer center, Phe¹⁵⁰.     

Theory of kinetic partitioning in protein folding with possible applications to prions

This study focuses on the phenomenon of kinetic partitioning when a polypeptide chain has two ground-state conformations, one of which is kinetically more reachable than the other. We designed sequences for lattice model proteins with two different conformations of equal energy corresponding to the global energy minimum. Folding simulations revealed that one of these conformations was indeed much more kinetically accessible than the other. We found that the number and strength of local contacts in the ground-state conformation are the major factors that determine which conformation is reached faster; the greater the number of local contacts, the more kinetically reachable a conformation is. We present simple statistical–mechanical arguments to explain these findings. Our results may be relevant in explaining the phenomenology of such proteins as human plasminogen activator inhibitor-1 (PAI-1), photosystem II, and prions. Proteins 31:335–344, 1998. © 1998 Wiley-Liss, Inc.     

Dual role of SnoN in mammalian tumorigenesis

SnoN is an important negative regulator of transforming growth factor beta signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. Here we show for the first time that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed pro-oncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both pro-tumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. Our study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression.     

The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.     

Analysis of the conformations corresponding to hexapeptide and large sequences characterized by continuous single amino acid repeats in proteins

The analysis of conformations corresponding to continuous amino acid repeat peptides (CARPs) comprising six or more residues in proteins of known three-dimensional structure revealed that alanine, glycine, glutamic acid, proline, valine, histidine, aspartic acid, glutamine and lysine were associated as repeating amino acid residues. Alanine, glycine and histidine CARPs were most common, although the histidine hexapeptide and large CARPs mainly correspond to affinity tags and are not part of the native protein sequence. The Ala and Glu CARPs were observed either as part of helix, or coil or a combination of these conformations. The octapeptide Ala CARP in six-hairpin glycosidases was observed as part of strand and coil conformation. The Gly and Pro CARPs were mainly associated with coil conformation. Majority of the coil regions in CARPs contained beta and gamma-turn structural motifs. The conformations of the Asp, Glu and Lys hexapeptide or larger CARPs were not defined in the corresponding protein three-dimensional structures analyzed. The longest CARP of known conformation was observed for alanine as a decapeptide in a lysozyme-like protein that corresponds to helix. A feature of CARPs is that a majority are exposed to solvent with accessible surface area greater than 200 ?(2) units in the protein three-dimensional structure.