Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.

Using the CATH domain database to assign structures and functions to the genome sequences.

The CATH database of protein structures contains approximately 18000 domains organized according to their (C)lass, (A)rchitecture, (T)opology and (H)omologous superfamily. Relationships between evolutionary related structures (homologues) within the database have been used to test the sensitivity of various sequence search methods in order to identify relatives in Genbank and other sequence databases. Subsequent application of the most sensitive and efficient algorithms, gapped blast and the profile based method, Position Specific Iterated Basic Local Alignment Tool (PSI-BLAST), could be used to assign structural data to between 22 and 36 % of microbial genomes in order to improve functional annotation and enhance understanding of biological mechanism. However, on a cautionary note, an analysis of functional conservation within fold groups and homologous superfamilies in the CATH database, revealed that whilst function was conserved in nearly 55% of enzyme families, function had diverged considerably, in some highly populated families. In these families, functional properties should be inherited far more cautiously and the probable effects of substitutions in key functional residues carefully assessed.

Diversity of conformational states and changes within the EF-hand protein superfamily.

The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca2+ binding, is found in a large number of functionally diverse Ca2+ binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca2+ to the open conformational state in its presence. It is now clear from structures of other EF-hand proteins that this "closed-to-open" conformational transition is not the sole model for EF-hand protein structural response to Ca2+. More complex modes of conformational change are observed in EF-hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF-hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF-hand proteins and its correlation with target recognition.

A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase.

The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy. In this complex, the CaMKK peptide forms a fold comprising an alpha-helix and a hairpin-like loop whose C-terminus folds back on itself. The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM-target complexes determined so far. The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively. This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes. The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins.

Genome analysis: Assigning protein coding regions to three-dimensional structures.

We describe the results of a procedure for maximizing the number of sequences that can be reliably linked to a protein of known three-dimensional structure. Unlike other methods, which try to increase sensitivity through the use of fold recognition software, we only use conventional sequence alignment tools, but apply them in a manner that significantly increases the number of relationships detected. We analyzed 11 genomes and found that, depending on the genome, between 23 and 32% of the ORFs had significant matches to proteins of known structure. In all cases, the aligned region consisted of either >100 residues or >50% of the smaller sequence. Slightly higher percentages could be attained if smaller motifs were also included. This is significantly higher than most previously reported methods, even those that have a fold-recognition component. We survey the biochemical and structural characteristics of the most frequently occurring proteins, and discuss the extent to which alignment methods can realistically assign function to gene products.

Combining sensitive database searches with multiple intermediates to detect distant homologues.

Using data from the CATH structure classification, we have assessed the blastp, fasta, smith-waterman and gapped-blast algorithms, developed a portable normalization scheme and identified safe thresholds for database searching. Of the four methods assessed, fasta, smith-waterman and gapped-blast perform similarly, whereas the sensitivity of blastp was much lower. Introduction of an intermediate sequence search substantially improved the results. When tested on a set of relationships that could not be identified by blastp, intermediate sequences were able to find double the number of relationships identified by the smith-waterman algorithm alone. However, we found that the benefit of using intermediates varied considerably between each family and depended not only on the number of available sequences, but also their diversity. In an attempt to increase sensitivity further, a multiple intermediate sequence search (MISS) procedure was developed. When assessed on 1906 cases from a wide range of homologous families that could not be detected by the previous approaches, MISS was able to identify 241 additional relationships. MISS uses the full extent of sequence diversity to detect additional relationships, but does not consider any structure-specific information. For this reason, it is more generally applicable than fold recognition and threading methods, which require a library of known structures.

NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ.

Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology. The histidine-aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region. The cytoplasmic region contains two functional domains: domain A (residues 223-289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290-450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heatshock protein 90 and DNA gyrase B.

NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: another example of an ancestral beta gamma-crystallin precursor structure.

The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Calpha, C') r.m.s.d. values of 0. 45(+/-0.11) A and 0.57(+/-0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two beta-sheets, each consisting of four antiparallel beta-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key beta-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain beta gamma-crystallin proteins, because of its structural similarity to each domain of the beta gamma-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the beta gamma-crystallin superfamily are believed to have evolved.

Assessing protein coding region integrity in cDNA sequencing projects.

MOTIVATION: In cDNA sequencing projects, it is vital to know whether the protein coding region of a sequence is complete, or whether errors have occurred during library construction. Here we present a linear discriminant approach that predicts this completeness by estimating the probability of each ATG being the initiation codon. RESULTS: Because of the current shortage of full-length cDNA data on which to base this work, tests were performed on a non-redundant set of 660 initiation codon-containing DNA sequences that had been conceptually spliced into mRNA/cDNA. We also used an edited set of the same sequences that only contained the region following the initiation codon as a negative control. Using the criterion that only a single prediction is allowed for each sequence, a cut-off was selected at which discrimination of both positive and negative sets was equal. At this cut-off, 67% of each set could be correctly distinguished, with the correct ATG codon also being identified in the positive set. Reliability could be increased further by raising the cut-off or including homologues, the relative merits of which are discussed. AVAILABILITY: The prediction program, called ATGpr, and other data are available at http://www.hri.co.jp/atgpr CONTACT: swintech@hri.co.jp

Solution structure of the IRF-2 DNA-binding domain: a novel subgroup of the winged helix-turn-helix family.

BACKGROUND: The transcription of interferon (IFN) and IFN-inducible genes is mainly regulated by the interferon regulatory factor (IRF) family of proteins, which recognize a unique AAGTGA hexamer repeat motif in the regulatory region of IFN genes. A DNA-binding domain of approximately 100 amino acids has been commonly found in the IRF family of proteins, but it has no sequence homology to known DNA-binding motifs. Elucidation of the structures of members of the IRF family is therefore useful to the understanding of the regulation and evolution of the immune system at the structural level. RESULTS: The solution structure of the DNA-binding domain of interferon regulatory factor-2 (IRF-2) has been determined by NMR spectroscopy. It is composed of a four-stranded antiparallel beta sheet and three alpha helices, and its global fold is similar to those of the winged helix-turn-helix (wHTH) family of proteins. A long loop (Pro37-Asp51) is found immediately before the HTH motif, which is not found in other wHTH proteins. The NMR signals of residues in this long loop, as well as the second helix of the HTH motif, are strongly affected upon the addition of the hexamer repeat DNA, suggesting that these structural elements participate in DNA recognition and binding. CONCLUSIONS: The structural similarity of the DNA-binding domain of IRF-2 with those of proteins in the wHTH family shows that the IRF proteins belong to the wHTH family, even though there is no apparent sequence homology among proteins of the two families. The sequential structure alignment program (SSAP) shows that IRF-2 has a slightly different structure from typical wHTH proteins, mainly in the orientation of helix 2. The IRF family of proteins should therefore be categorized into a subfamily of the wHTH family. The evidence here implies that the evolutional pathway of the IRF family is distinct from that of the other wHTH proteins, in other words, the immune system diverged from an evolutional stem at an early stage.

Domain assignment for protein structures using a consensus approach: characterization and analysis.

A consensus approach for the assignment of structural domains in proteins is presented. The approach combines a number of previously published algorithms, and takes advantage of the elevated accuracy obtained when assignments from the individual algorithms are in agreement. The consensus approach is tested on a data set of 55 protein chains, for which domain assignments from four automated methods were known, and for which crystallographers assignments had been reported in the literature. Accuracy was found to increase in this test from 72% using individual algorithms to 100% when all four methods were in agreement. However a consensus prediction using all four methods was only possible for 52% of the dataset. The consensus approach [using three publicly available domain assignment algorithms (PUU, DETECTIVE, DOMAK)] was then used to make domain assignments for a data set of 787 protein chains from the Protein Data Bank. Analysis of the assignments showed 55.7% of assignments could be made automatically, and of these, 13.5% were multi-domain proteins. Of the remaining 44.3% that could not be assigned by the consensus procedure 90.4% had their domain boundaries assigned correctly by at least one of the algorithms. Once identified, these domains were analyzed for trends in their size and secondary structure class. In addition, the discontinuity of each domain along the protein chain was considered.

Solution structure of calmodulin-W-7 complex: the basis of diversity in molecular recognition.

The solution structure of calcium-bound calmodulin (CaM) complexed with an antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), has been determined by multidimensional NMR spectroscopy. The structure consists of one molecule of W-7 binding to each of the two domains of CaM. In each domain, the W-7 chloronaphthalene ring interacts with four methionine methyl groups and other aliphatic or aromatic side-chains in a deep hydrophobic pocket, the site responsible for CaM binding to CaM-dependent enzymes such as myosin light chain kinases (MLCKs) and CaM kinase II. This competitive binding at the same site between W-7 and CaM-dependent enzymes suggests the mechanism by which W-7 inhibits CaM to activate the enzymes. The orientation of the W-7 naphthalene ring in the N-terminal pocket is rotated approximately 40 degrees with respect to that in the C-terminal pocket. The W-7 ring orientation differs significantly from the Trp800 indole ring of smooth muscle MLCK bound to the C-terminal pocket and the phenothiazine ring of trifluoperazine bound to the N or C-terminal pocket. These comparative structural analyses demonstrate that the two hydrophobic pockets of CaM can accommodate a variety of bulky aromatic rings, which provides a plausible structural basis for the diversity in CaM-mediated molecular recognition.

Contemporary approaches to protein structure classification.

In a similar manner to sequence database searching, it is also possible to compare three-dimensional protein structure. Such methods can be extremely useful because a structural similarity may represent a distant evolutionary relationship that is undetectable by sequence analysis. In this review, we summarise the most popular structure comparison methods, show how they can be used for database searching, and then describe some of the most advanced attempts to develop comprehensive protein structure classifications. With such data, it is possible to identify distant evolutionary relationships, provide libraries of unique folds for structure prediction, estimate the total number of folds that exist, and investigate the preference for certain types of structures over others.

Classifying a protein in the CATH database of domain structures.

The CATH database of protein domain structures classifies structures according to their (C)lass, (A)rchitecture, (T)opology or fold and (H)omologous family (http://www.biochem.ucl.ac.uk/bsm/cath). Although the protocol used is mostly automatic, manual inspection is used to check assignments at some critical stages, such as the detection of very distantly related homologues and anologues and the assignment of novel architectures. Described in this article is a recently established facility to search the database with the coordinates of a newly determined structure. The CATH server first locates domain boundaries and then uses automatic sequence and structure comparison methods to assign this new structure to one or more of the domain families within CATH. Diagnostic reports are generated, together with multiple structural alignments for close relatives. The Server can be accessed over the World Wide Web (WWW) and mirror sites are planned to improve access.

CATH--a hierarchic classification of protein domain structures.

BACKGROUND: Protein evolution gives rise to families of structurally related proteins, within which sequence identities can be extremely low. As a result, structure-based classifications can be effective at identifying unanticipated relationships in known structures and in optimal cases function can also be assigned. The ever increasing number of known protein structures is too large to classify all proteins manually, therefore, automatic methods are needed for fast evaluation of protein structures. RESULTS: We present a semi-automatic procedure for deriving a novel hierarchical classification of protein domain structures (CATH). The four main levels of our classification are protein class (C), architecture (A), topology (T) and homologous superfamily (H). Class is the simplest level, and it essentially describes the secondary structure composition of each domain. In contrast, architecture summarises the shape revealed by the orientations of the secondary structure units, such as barrels and sandwiches. At the topology level, sequential connectivity is considered, such that members of the same architecture might have quite different topologies. When structures belonging to the same T-level have suitably high similarities combined with similar functions, the proteins are assumed to be evolutionarily related and put into the same homologous superfamily. CONCLUSIONS: Analysis of the structural families generated by CATH reveals the prominent features of protein structure space. We find that nearly a third of the homologous superfamilies (H-levels) belong to ten major T-levels, which we call superfolds, and furthermore that nearly two-thirds of these H-levels cluster into nine simple architectures. A database of well-characterised protein structure families, such as CATH, will facilitate the assignment of structure-function/evolution relationships to both known and newly determined protein structures.

Protein clefts in molecular recognition and function.

One of the primary factors determining how proteins interact with other molecules is the size of clefts in the protein's surface. In enzymes, for example, the active site is often characterized by a particularly large and deep cleft, while interactions between the molecules of a protein dimer tend to involve approximately planar surfaces. Here we present an analysis of how cleft volumes in proteins relate to their molecular interactions and functions. Three separate datasets are used, representing enzyme-ligand binding, protein-protein dimerization and antibody-antigen complexes. We find that, in single-chain enzymes, the ligand is bound in the largest cleft in over 83% of the proteins. Usually the largest cleft is considerably larger than the others, suggesting that size is a functional requirement. Thus, in many cases, the likely active sites of an enzyme can be identified using purely geometrical criteria alone. In other cases, where there is no predominantly large cleft, chemical interactions are required for pinpointing the correct location. In antibody-antigen interactions the antibody usually presents a large cleft for antigen binding. In contrast, protein-protein interactions in homodimers are characterized by approximately planar interfaces with several clefts involved. However, the largest cleft in each subunit still tends to be involved.

Structural similarity between the pleckstrin homology domain and verotoxin: the problem of measuring and evaluating structural similarity.

An unexpected structural similarity is described between the pleckstrin homology (PH) domain and verotoxin. This similarity has escaped detection primarily due to the differences in topology that exist between the two proteins. By comparing this result with two previously reported similarities for the PH domain, one with the lipocalins and another with the FK506 binding protein, we discuss the problems of measuring and assessing structural similarities.