Solution NMR study of the monomeric form of p13suc1 protein sheds light on the hinge region determining the affinity for a phosphorylated substrate.

Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal beta-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13(suc1) CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the beta-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13(suc1) and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal beta-sheet involved in domain swapping and protein interactions.     

Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1: implications for domain swapping, anion binding and protein interactions

BACKGROUND: The Saccharomyces cerevisiae protein Cks1 (cyclin-dependent kinase subunit 1) is essential for cell-cycle progression. The biological function of Cks1 can be modulated by a switch between two distinct molecular assemblies: the single domain fold, which results from the closing of a beta-hinge motif, and the intersubunit beta-strand interchanged dimer, which arises from the opening of the beta-hinge motif. The crystal structure of a cyclin-dependent kinase (Cdk) in complex with the human Cks homolog CksHs1 single-domain fold revealed the importance of conserved hydrophobic residues and charged residues within the beta-hinge motif. RESULTS: The 3.0 A resolution Cks1 structure reveals the strict structural conservation of the Cks alpha/beta-core fold and the beta-hinge motif. The beta hinge identified in the Cks1 structure includes a novel pivot and exposes a cluster of conserved tyrosine residues that are involved in Cdk binding but are sequestered in the beta-interchanged Cks homolog suc1 dimer structure. This Cks1 structure confirms the conservation of the Cks anion-binding site, which interacts with sidechain residues from the C-terminal alpha helix of another subunit in the crystal. CONCLUSIONS: The Cks1 structure exemplifies the conservation of the beta-interchanged dimer and the anion-binding site in evolutionarily distant yeast and human Cks homologs. Mutational analyses including in vivo rescue of CKS1 disruption support the dual functional roles of the beta-hinge residue Glu94, which participates in Cdk binding, and of the anion-binding pocket that is located 22 A away and on an opposite face to Glu94. The Cks1 structure suggests a biological role for the beta-interchanged dimer and the anion-binding site in targeting Cdks to specific phosphoproteins during cell-cycle progression.     

Surface topography of the p3 and p6 annexin V crystal forms determined by atomic force microscopy

Annexin V is a member of a family of structurally homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. The structure of the soluble form of annexin V has been solved by X-ray crystallography, while electron crystallography of two-dimensional (2D) crystals has been used to reveal the structure of its membrane-bound form. Two 2D crystal forms of annexin V have been reported to date, with either p6 or p3 symmetry. Atomic force microscopy has previously been used to investigate the growth and the topography of the p6 crystal form on supported phospholipid bilayers (Reviakine et al., 1998). The surface structure of the second crystal form, p3, is presented in this study, along with an improved topographic map of the p6 crystal form. The observed topography is correlated with the structure determined by X-ray crystallography.     

Interaction between the cell-cycle-control proteins p34cdc2 and p9CKShs2. Evidence for two cooperative binding domains in p9CKShs2.

A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone H1 kinase activity and constituted of at least two subunits, p34cdc2 and cyclin Bcdc13, triggers the G2----M transition of the cell cycle in all organisms. The yeast p13suc1 and p18CKS1 subunits and their functionally interchangeable human homologues, p9CKShs1 and p9CKShs2, directly interact with p34cdc2 and may actually be part of the MPF complex. We have chemically synthesized p9CKShs2 and several of its peptide domains in order to investigate the binding of p9CKShs2 and p34cdc2. Several arguments support the hypothesis that the N-terminal half (peptide B) and the C-terminal half (peptide E) each contain a p34cdc2-binding site and that these two binding domains cooperate in establishing a stable p9CKShs2-p34cdc2 complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKShs1-Sepharose beads; (b) only immobilized peptides B + E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs1 for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B- or E-Sepharose beads, respectively. No binding occurs in the absence of free peptide. This additivity cannot be attributed to the formation of a B-E complex mimicking the full-length p9CKShs2. The cyclin B subunit is not required for the formation of the p9CKShs2-p34cdc2 complex through these two binding domains. The implications of the existence of two cooperative p34cdc2-binding domains in p9CKShs2 on the structure of the active M-phase-specific kinase is discussed.     

An extensively associated dimer in the structure of the C713S mutant of the TIR domain of human TLR2

The Toll/interleukin-1 receptor (TIR) domains are conserved modules in the intracellular regions of the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). The domains are crucial for the signal transduction by these receptors, through homotypic interactions among the receptor and the downstream adapter TIR domains. Previous studies showed that the BB loop in the structure of the TIR domain forms a prominent conserved feature on the surface and is important for receptor signaling. Here we report the crystal structure of the C713S mutant of the TIR domain of human TLR2. An extensively associated dimer is observed in the crystal structure and mutations of several residues in this dimer interface abolished the function of the receptor. Moreover, the structure shows that the BB loop can adopt different conformations, which are required for the formation of this dimer. This asymmetric dimer might represent the TLR2:TLRx heterodimer in the function of this receptor.     

1.9 A resolution crystal structure of the Saccharomyces cerevisiae Ran-binding protein Mog1p

The 1.9 A resolution X-ray crystal structure of Ran-binding protein Mog1p shows that it has a unique fold based on a six-stranded antiparallel beta-sheet backed on both sides by an extensive alpha-helix. The topology of some elements of Mog1p secondary structure resemble a portion of nuclear transport factor 2 (NTF2), but the hydrophobic cavity and surrounding negatively charged residues that are important in the NTF2-RanGDP interaction are not conserved in Mog1p. In addition to binding RanGTP, Mog1p forms a 1:1 complex with RanGDP and so binds Ran independent of its nucleotide state. Mog1p and NTF2 compete for binding to RanGDP indicating that their binding sites on RanGDP are sufficiently close to prevent both proteins binding simultaneously. Although there may be some overlap between the Mog1p and NTF2 binding sites on RanGDP, these sites are not identical. Sequence analysis of Mog1p homologues from Schizosaccharomyces pombe, human, and Caenorhabditis elegans in the context of the Mog1p crystal structure indicates the presence of a cluster of highly conserved surface residues consistent with an interaction site for Ran.     

The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites

The mRNA of Escherichia coli yedU gene is induced 31-fold upon heat shock. The 31-kD YedU protein, also calls Hsp31, is highly conserved in several human pathogens and has chaperone activity. We solved the crystal structure of YedU at 2.2 A resolution. YedU monomer has an alpha/beta/alpha sandwich domain and a small alpha/beta domain. YedU is a dimer in solution, and its crystal structure indicates that a significant amount of surface area is buried upon dimerization. There is an extended hydrophobic patch that crosses the dimer interface on the surface of the protein. This hydrophobic patch is likely the substrate-binding site responsible for the chaperone activity. The structure also reveals a potential protease-like catalytic triad composed of Cys184, His185, and Asp213, although no enzymatic activity could be identified. YedU coordinates a metal ion using His85, His122, and Glu90. This 2-His-1-carboxylate motif is present in carboxypeptidase A (a zinc enzyme), and a number of dioxygenases and hydroxylases that utilize iron as a cofactor, suggesting another potential function for YedU.     

Observation of signal transduction in three-dimensional domain swapping

p13suc1 (suc1) has two native states, a monomer and a domain-swapped dimer. The structure of each subunit in the dimer is identical to that of the monomer, except for the hinge loop that connects the exchanging domains. Here we find that single point mutations at sites throughout the protein and ligand binding both shift the position of the equilibrium between monomer and dimer. The hinge loop was shown previously to act as a loaded molecular spring that releases tension present in the monomer by adopting an alternative conformation in the dimer. The results here indicate that the release of strain propagates throughout the entire protein and alters the energetics of regions remote from the hinge. Our data illustrate how the signal conferred by the conformational change of a protein loop, elicited by domain swapping, ligand binding or mutation, can be sensed by a distant active site. This work highlights the potential role of strained loops in proteins: the energy they store can be used for both signal transduction and allostery, and they could steer the evolution of protein function. Finally, a structural mechanism for the role of suc1 as an adapter molecule is proposed.     

Characterization of the unfolding pathway of the cell-cycle protein p13suc1 by molecular dynamics simulations: implications for domain swapping

BACKGROUND: The p13suc1 gene product is a member of the cks (cyclin-dependent protein kinase subunit) protein family and has been implicated in regulation of the cell cycle. Various crystal structures of suc1 are available, including a globular, monomeric form and a beta-strand exchanged dimer. It has been suggested that conversions between these forms, and perhaps others, may be important in the regulation of the cell cycle. RESULTS: We have undertaken molecular dynamics simulations of protein unfolding to investigate the conformational properties of suc1. Unfolding transition states were identified for each of four simulations. These states contain some native secondary structure, primarily helix alpha1 and the core of the beta sheet. The hydrophobic core is loosely packed. Further unfolding leads to an intermediate state that is slightly more expanded than the transition state, but with considerably fewer nonlocal, tertiary packing contacts and less secondary structure. The helices are fluctuating but partially formed in the denatured state and beta2 and beta4 remain associated. CONCLUSIONS: It appears that suc1 folds by a nucleation-condensation mechanism, similar to that observed for two-state folding proteins. However, suc1 forms an intermediate during unfolding and contains considerable residual structure in the denatured state. The stability of the beta2-beta4 residual structure is surprising, because beta4 is the strand involved in domain swapping. This stability suggests that the domain-swapping event, if physiologically relevant, may require the assistance of additional factors in vivo or occur early in the folding process.     

Crystal structure of the post-chaperonin beta-tubulin binding cofactor Rbl2p.

The folding pathway of tubulins includes highly specific interactions with a series of cofactors (A, B, C, D and E) after they are released from the eukaryotic chaperonin CCT. The 2.2 A crystal structure of Rbl2p, the Saccharomyces cerevisiae homolog of beta-tubulin specific cofactor A, shows alpha-helical monomers forming a flat, slightly convex dimer. The surface of the molecule is dominated by polar and charged residues and lacks hydrophobic patches typically observed for chaperones that bind unfolded or partially folded proteins. This post-chaperonin cofactor is therefore clearly distinct from typical chaperones where hydrophobicity is a hallmark of substrate recognition.     

Crystal structure of archaeal ribonuclease P protein Ph1771p from Pyrococcus horikoshii OT3: an archaeal homolog of eukaryotic ribonuclease P protein Rpp29

Ribonuclease P (RNase P) is the endonuclease responsible for the removal of 5' leader sequences from tRNA precursors. The crystal structure of an archaeal RNase P protein, Ph1771p (residues 36-127) from hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined at 2.0 A resolution by X-ray crystallography. The structure is composed of four helices (alpha1-alpha4) and a six-stranded antiparallel beta-sheet (beta1-beta6) with a protruding beta-strand (beta7) at the C-terminal region. The strand beta7 forms an antiparallel beta-sheet by interacting with strand beta4 in a symmetry-related molecule, suggesting that strands beta4 and beta7 could be involved in protein-protein interactions with other RNase P proteins. Structural comparison showed that the beta-barrel structure of Ph1771p has a topological resemblance to those of Staphylococcus aureus translational regulator Hfq and Haloarcula marismortui ribosomal protein L21E, suggesting that these RNA binding proteins have a common ancestor and then diverged to specifically bind to their cognate RNAs. The structure analysis as well as structural comparison suggested two possible RNA binding sites in Ph1771p, one being a concave surface formed by terminal alpha-helices (alpha1-alpha4) and beta-strand beta6, where positively charged residues are clustered. A second possible RNA binding site is at a loop region connecting strands beta2 and beta3, where conserved hydrophilic residues are exposed to the solvent and interact specifically with sulfate ion. These two potential sites for RNA binding are located in close proximity. The crystal structure of Ph1771p provides insight into the structure and function relationships of archaeal and eukaryotic RNase P.     

Solution structure of the lymphocyte receptor Nkrp1a reveals a distinct conformation of the long loop region as compared to in the crystal structure

Mouse Nkrp1a receptor is a C‐type lectin‐like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined. A notable difference between the crystal and NMR structure of Nkrp1a appears in the conformation of the long loop region. While the extended loop points away from the central core and mediates formation of a domain swapped dimer in the crystal, the solution structure is monomeric with the loop tightly anchored to the central region. The findings described the first solution structure in the Nkrp1 family and revealed intriguing similarities and differences to the crystal structure. Proteins 2016; 84:1304–1311. © 2016 Wiley Periodicals, Inc.     

Platelet-activating factor- and thrombin-induced stimulation of p34cdc2-cyclin histone H1 kinase activity in platelets.

Numerous studies of cell cycle control in dividing cells have pointed to the central role of a 34-kDa histone H1 kinase (p34cdc2) complexed with regulatory subunits known as cyclins. We now report that p34cdc2-cyclin may also participate in signal transduction in nonproliferating, terminally differentiated cells, in this instance during sheep platelet activation. Immunological evidence for the presence of a p34cdc2 cognant in sheep platelet cytosol was obtained with antipeptide antibodies raised against peptide sequences in the conserved PSTAIRE and C-terminus regions of murine cdc2. The immunoreactive 32-kDa protein was adsorbed onto p13suc1-Sepharose, which selectively binds p34cdc2. A 58-kDa protein that also bound to p13suc1-Sepharose was identified as cyclin A on the basis of its size and immunoreactivity with two different anticyclin peptide antibodies. The p34cdc2-cyclin A complex was regulated during platelet activation. Its histone H1 phosphorylating activity was stimulated 2-fold in p13suc1-Sepharose extracts from platelets that had been exposed to platelet-activating factor or thrombin for 1 min prior to harvesting. Our findings imply that the p34cdc2-cyclin complex may serve alternative functions besides control of cell division.     

p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro.

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 M NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.     

The role of the Tim8p-Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p-Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p-Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p-Tim13p complex is not dependent on zinc, and the purified Tim8p-Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.     

Solution structure of ribosomal protein L40E, a unique C4 zinc finger protein encoded by archaeon Sulfolobus solfataricus

The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X(10)_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin-fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three-dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three-stranded beta-sheet with a simple beta2beta1beta3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered beta2-beta3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition.     

Crystal structure of ferric-yersiniabactin, a virulence factor of Yersinia pestis

Yersiniabactin (Ybt), the siderophore produced by Yersinia pestis, has been crystallized successfully in the ferric complex form and the crystal structure has been determined. The crystals are orthorhombic with a space group of P2(1)2(1)2(1) and four distinct molecules per unit cell with cell dimensions of a=11.3271(+/-0.0003)A, b=22.3556(+/-0.0006)A, and c=39.8991(+/-0.0011)A. The crystal structure of ferric Ybt shows that the ferric ion is coordinated as a 1:1 complex by three nitrogen electron pairs and three negatively charged oxygen atoms with a distorted octahedral coordination. The molecule displays a Delta absolute configuration with chiral centers at N2, C9, C10, C12, C13, and C19 in R, R, R, R, S, S configurations, respectively. Few of the crystal structures of siderophores have been solved, and those which have been are of simple hydroxamate and catechol types such as ferrioxamine B and agrobactin. To our knowledge this is the first report of the ferric crystal structure of 5-member heterocycle siderophore.     

Solution structure of Phl p 3, a major allergen from timothy grass pollen

The major 97-aa timothy grass (Phleum pratense) allergen Phl p 3 was recently isolated from an extract of timothy grass pollen. Sequence comparison classifies this protein as a group 3 allergen. The solution structure of Phl p 3 as determined by nuclear magnetic resonance spectroscopy reveals that the protein consists of a core of hydrophobic amino-acid side chains from two beta-sheets of five and four anti-parallel beta-strands, respectively. This conformation is very similar to the crystal structure published for Phl p 2 and strongly resembles the known conformation of the carboxy-terminal domain of Phl p 1, the major difference being the loop orientations. Phl p 2 and Phl p 3 show virtually identical immunoreactivity, and comparison of the charged surface amino acids of the two proteins gives initial clues as to the IgE recognition epitopes of these proteins.