Sequence and structure continuity of evolutionary importance improves protein functional site discovery and annotation.

Protein functional sites control most biological processes and are important targets for drug design and protein engineering. To characterize them, the evolutionary trace (ET) ranks the relative importance of residues according to their evolutionary variations. Generally, top-ranked residues cluster spatially to define evolutionary hotspots that predict functional sites in structures. Here, various functions that measure the physical continuity of ET ranks among neighboring residues in the structure, or in the sequence, are shown to inform sequence selection and to improve functional site resolution. This is shown first, in 110 proteins, for which the overlap between top-ranked residues and actual functional sites rose by 8% in significance. Then, on a structural proteomic scale, optimized ET led to better 3D structure-function motifs (3D templates) and, in turn, to enzyme function prediction by the Evolutionary Trace Annotation (ETA) method with better sensitivity of (40% to 53%) and positive predictive value (93% to 94%). This suggests that the similarity of evolutionary importance among neighboring residues in the sequence and in the structure is a universal feature of protein evolution. In practice, this yields a tool for optimizing sequence selections for comparative analysis and, via ET, for better predictions of functional site and function. This should prove useful for the efficient mutational redesign of protein function and for pharmaceutical targeting.

Background frequencies for residue variability estimates: BLOSUM revisited.

BACKGROUND: Shannon entropy applied to columns of multiple sequence alignments as a score of residue conservation has proven one of the most fruitful ideas in bioinformatics. This straightforward and intuitively appealing measure clearly shows the regions of a protein under increased evolutionary pressure, highlighting their functional importance. The inability of the column entropy to differentiate between residue types, however, limits its resolution power. RESULTS: In this work we suggest generalizing Shannon's expression to a function with similar mathematical properties, that, at the same time, includes observed propensities of residue types to mutate to each other. To do that, we revisit the original construction of BLOSUM matrices, and re-interpret them as mutation probability matrices. These probabilities are then used as background frequencies in the revised residue conservation measure. CONCLUSION: We show that joint entropy with BLOSUM-proportional probabilities as a reference distribution enables detection of protein functional sites comparable in quality to a time-costly maximum-likelihood evolution simulation method (rate4site), and offers greater resolution than the Shannon entropy alone, in particular in the cases when the available sequences are of narrow evolutionary scope.

On itinerant water molecules and detectability of protein-protein interfaces through comparative analysis of homologues.

We discuss the question of which residues are sufficiently important for protein-protein interaction to be under notable evolutionary pressure. Its interest stems from the applicability of this knowledge in the reverse direction, to detect a protein-protein interface on a single protomer, starting from the rate of mutation of participating residues. Using the analysis of trajectories produced by the molecular dynamics simulations, we suggest that, in the case of water soluble proteins, a large fraction of evolutionarily privileged residues can be found by considering the dynamic behavior of the protein interface and by looking for residues which exchange water molecules with the bulk of the solvent outstandingly slowly (tentatively termed "dry residues"). We show that the dry interface residues are better conserved across homologues than the generic "geometric footprint" and can be quite reliably detected through comparative analysis of protein homologues, without strong dependence on the choice of method. Furthermore, we show that dry residues distinguish themselves through a set of biophysical properties consistent with the known mechanisms of protein oligomerization: their compositional shift toward nonpolar, overlap, and co-location with residues exhibiting low mobility, their two- to threefold increased propensity over the rest of the geometric footprint to form hydrogen bonds, and four- to almost tenfold increased likelihood to participate in formation of salt bridges. These properties, consistently, help understand the observed increase in the evolutionary pressure that dry residues experience.

Evolutionary trace report_maker: a new type of service for comparative analysis of proteins.

: Evolutionary trace report_maker offers a new type of service for researchers investigating the function of novel proteins. It pools, from different sources, information about protein sequence, structure and elementary annotation, and to that background superimposes inference about the evolutionary behavior of individual residues, using real-valued evolutionary trace method. As its only input it takes a Protein Data Bank identifier or UniProt accession number, and returns a human-readable document in PDF format, supplemented by the original data needed to reproduce the results quoted in the report.

Evolutionary and structural feedback on selection of sequences for comparative analysis of proteins.

It has been noted that slowly evolving protein residues have two properties: (a) they tend to cluster in the native fold, and (b) they delineate functional surfaces-parts of the surface through which the protein interacts with other proteins or small ligands. Herein, we demonstrate that the two are coupled sufficiently strongly that one effect, when observed, statistically implies the other. Detection of both can be accomplished in multiple sequence alignment related methods by the careful selection of relevant sequences. For the demonstration, we use two sets of protein families: a small set of diverse proteins with diverse functional surfaces, and a large set of homodimerizing enzymes. A practical outcome of our considerations is a simple prescriptive rule for the selection of homologous sequences for the comparative analysis of proteins: in order to optimize the detection of (potentially unknown) functional surfaces, it is sufficient to select sequences in such a way that the residues observed at any level of evolutionary divergence, as implied by the alignment, cluster on the folded protein.

A structure and evolution-guided Monte Carlo sequence selection strategy for multiple alignment-based analysis of proteins.

MOTIVATION: Various multiple sequence alignment-based methods have been proposed to detect functional surfaces in proteins, such as active sites or protein interfaces. The effect that the choice of sequences has on the conclusions of such analysis has seldom been discussed. In particular, no method has been discussed in terms of its ability to optimize the sequence selection for the reliable detection of functional surfaces. RESULTS: Here we propose, for the case of proteins with known structure, a heuristic Metropolis Monte Carlo strategy to select sequences from a large set of homologues, in order to improve detection of functional surfaces. The quantity guiding the optimization is the clustering of residues which are under increased evolutionary pressure, according to the sample of sequences under consideration. We show that we can either improve the overlap of our prediction with known functional surfaces in comparison with the sequence similarity criteria of selection or match the quality of prediction obtained through more elaborate non-structure based-methods of sequence selection. For the purpose of demonstration we use a set of 50 homodimerizing enzymes which were co-crystallized with their substrates and cofactors.

An evolution based classifier for prediction of protein interfaces without using protein structures.

MOTIVATION: The number of available protein structures still lags far behind the number of known protein sequences. This makes it important to predict which residues participate in protein-protein interactions using only sequence information. Few studies have tackled this problem until now. RESULTS: We applied support vector machines to sequences in order to generate a classification of all protein residues into those that are part of a protein interface and those that are not. For the first time evolutionary information was used as one of the attributes and this inclusion of evolutionary importance rankings improves the classification. Leave-one-out cross-validation experiments show that prediction accuracy reaches 64%.

A family of evolution-entropy hybrid methods for ranking protein residues by importance.

In order to identify the amino acids that determine protein structure and function it is useful to rank them by their relative importance. Previous approaches belong to two groups; those that rely on statistical inference, and those that focus on phylogenetic analysis. Here, we introduce a class of hybrid methods that combine evolutionary and entropic information from multiple sequence alignments. A detailed analysis in insulin receptor kinase domain and tests on proteins that are well-characterized experimentally show the hybrids' greater robustness with respect to the input choice of sequences, as well as improved sensitivity and specificity of prediction. This is a further step toward proteome scale analysis of protein structure and function.

Combining inference from evolution and geometric probability in protein structure evaluation.

Starting from the hypothesis that evolutionarily important residues form a spatially limited cluster in a protein's native fold, we discuss the possibility of detecting a non-native structure based on the absence of such clustering. The relevant residues are determined using the Evolutionary Trace method. We propose a quantity to measure clustering of the selected residues on the structure and show that the exact values for its average and variance over several ensembles of interest can be found. This enables us to study the behavior of the associated z-scores. Since our approach rests on an analytic result, it proves to be general, customizable, and computationally fast. We find that clustering is indeed detectable in a large representative protein set. Furthermore, we show that non-native structures tend to achieve lower residue-clustering z-scores than those attained by the native folds. The most important conclusion that we draw from this work is that consistency between structural and evolutionary information, manifested in clustering of key residues, imposes powerful constraints on the conformational space of a protein.

Prediction and confirmation of a site critical for effector regulation of RGS domain activity.

A critical challenge of structural genomics is to extract functional information from protein structures. We present an example of how this may be accomplished using the Evolutionary Trace (ET) method in the context of the regulators of G protein signaling (RGS) family. We have previously applied ET to the RGS family and identified a novel, evolutionarily privileged site on the RGS domain as important for regulating RGS activity. Here we confirm through targeted mutagenesis of RGS7 that these ET-identified residues are critical for RGS domain regulation and are likely to function as global determinants of RGS function. We also discuss how the recent structure of the complex of RGS9, Gt/i1alpha-GDP-AlF4- and the effector subunit PDEgamma confirms their contact with the effector-G protein interface, forming a structural pathway that communicates from the effector-contacting surface of the G protein and RGS catalytic core domain to the catalytic interface between Galpha and RGS. These results demonstrate the effectiveness of ET for identifying binding sites and efficiently focusing mutational studies on their key residues, thereby linking raw sequence and structure data to functional information.

Genetic mapping of the human C5a receptor. Identification of transmembrane amino acids critical for receptor function.

Many hormones and sensory stimuli signal through a superfamily of seven transmembrane-spanning receptors to activate heterotrimeric G proteins. How the seven transmembrane segments of the receptors (a molecular architecture of bundled alpha-helices conserved from yeast to man) work as "on/off" switches remains unknown. Previously, we used random saturation mutagenesis coupled with a genetic selection in yeast to determine the relative importance of amino acids in four of the seven transmembrane segments of the human C5a receptor (Baranski, T. J., Herzmark, P., Lichtarge, O., Gerber, B. O., Trueheart, J., Meng, E. C., Iiri, T., Sheikh, S. P., and Bourne, H. R. (1999) J. Biol. Chem. 274, 15757-15765). In this study, we evaluate helices I, II, and IV, thereby furnishing a complete mutational map of the seven transmembrane helices of the human C5a receptor. Our analysis identified 19 amino acid positions resistant to non-conservative substitutions. When combined with the 25 essential residues previously identified in helices III and V-VII, they delineate two distinct components of the receptor switch: a ligand-binding surface at or near the extracellular surface of the helix bundle and a core cluster in the cytoplasmic half of the bundle. In addition, we found critical amino acids in the first and second helices that are predicted to face the lipid membrane. These residues form an extended surface that might mediate interactions with lipids and other membrane proteins or function as an oligomerization domain with other receptors.

A genome-wide search for synaptic vesicle cycle proteins in Drosophila.

The recent completion of the Drosophila genome sequence opens new avenues for neurobiology research. We screened the fly genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis and endocytosis of synaptic vesicles. We identified fly homologs for 93% of the vertebrate genes that were screened. These are on average 60% identical and 74% similar to their vertebrate counterparts. This high degree of conservation suggests that little protein diversification has been tolerated in the evolution of synaptic transmission. Finally, and perhaps most exciting for Drosophila neurobiologists, the genomic sequence allows us to identify P element transposon insertions in or near genes, thereby allowing rapid isolation of mutations in genes of interest. Analysis of the phenotypes of these mutants should accelerate our understanding of the role of numerous proteins implicated in synaptic transmission.

Influence of mutation type and X chromosome inactivation on Rett syndrome phenotypes.

We screened 71 sporadic and 7 familial Rett syndrome (RTT) patients for MECP2 mutations by direct sequencing and determined the pattern of X chromosome inactivation (XCI) in 39 RTT patients. We identified 23 different disease-causing MECP2 mutations in 54 of 71 (76%) sporadic patients and in 2 of 7 (29%) familial cases. We compared electrophysiological findings, cerebrospinal fluid neurochemistry, and 13 clinical characteristics between patients carrying missense mutations and those carrying truncating mutations. Thirty-one of 34 patients (91%) with classic RTT had random XCI. Nonrandom XCI was associated with milder phenotypes, including a mitigated classic RTT caused by a rare early truncating mutation. Patients with truncating mutations have a higher incidence of awake respiratory dysfunction and lower levels of cerebrospinal fluid homovanillic acid. Scoliosis is more common in patients with missense mutations. These data indicate that different MECP2 mutations have similar phenotypic consequences, and random XCI plays an important role in producing the full phenotypic spectrum of classic RTT. The association of early truncating mutations with nonrandom XCI, along with the fact that chimeric mice lacking methyl-CpG-binding protein 2 (MeCP2) function die during embryogenesis, supports the notion that RTT is caused by partial loss of MeCP2 function.

A regulator of G protein signaling interaction surface linked to effector specificity.

Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the alpha subunits (G(alpha)) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual G(alpha), and GTP hydrolysis rates of different G(alpha)s can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-G(alpha) binding interface and extends to include additional functionally important residues on the surface. One of these is within helix alpha3, two are in alpha5, and three are in the loop connecting alpha5 and alpha6. A cluster of surface residues on G(alpha) previously identified by ET, and composed predominantly of residues from the switch III region and helix alpha3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the gamma subunit of G(talpha)'s effector, cGMP phosphodiesterase (PDEgamma). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-G(alpha) complex. Sequence variations in these residues correlate with PDEgamma effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions.

Similar structures and shared switch mechanisms of the beta2-adrenoceptor and the parathyroid hormone receptor. Zn(II) bridges between helices III and VI block activation.

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A. , Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the beta2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.

C5a receptor activation. Genetic identification of critical residues in four transmembrane helices.

Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.

The murine Polycomb-group gene eed and its human orthologue: functional implications of evolutionary conservation.

Similar to Drosophila, murine Polycomb-group (PcG) genes regulate anterior-posterior patterning of segmented axial structures by transcriptional repression of homeotic gene expression. The murine PcG gene eed (embryonic ectoderm development) encodes a 441-amino-acid protein with five WD motifs which, except for the amino terminus, is highly homologous to Drosophila ESC (Extra Sex Combs). Here, sequence and expression analysis as well as chromosomal mapping of the human orthologue of eed is described. Absolute conservation of the human eed protein along with significant divergence at the nucleotide level reveals functional constraints operating on all residues. The human orthologue appears to be ubiquitously expressed and maps to chromsome 11q14.2-q22.3. Using the first WD motif of the beta-subunit of the bovine G protein as a structural reference, the predicted locations of two previously identified eed point mutations (A. Schumacher et al., 1996, Nature 383: 250-253) are also reported herein. The proline substitution (L196P) in the second WD motif of the l7Rn5(3354SB) null allele maps to the internal core of the inner end of the beta-propeller blade and is likely to disrupt protein folding. In contrast, the asparagine substitution (I193N) in the second WD motif of the hypomorphic l7Rn5(1989SB) allele maps onto the surface of the beta-propeller blade near the central cavity and may affect surface interactions without compromising propeller packing. These results illustrate the critical importance of all residues for eed function in mammals and support a model whereby the amino terminus might implement function(s) related to embryonic development in higher organisms.

Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin.

Transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have revealed contact surfaces between G protein subunits, but not the surfaces or molecular mechanism through which Galphabetagamma responds to activation by transmembrane receptors. Such a surface was identified from the results of testing 100 mutant alpha subunits of the retinal G protein transducin for their ability to interact with rhodopsin. Sites at which alanine substitutions impaired this interaction mapped to two distinct Galpha surfaces: a betagamma-binding surface and a putative receptor-interacting surface. On the basis of these results a mechanism for receptor-catalyzed exchange of guanosine diphosphate for guanosine triphosphate is proposed.

Meeting review: the Second meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2), Asilomar, California, December 13-16, 1996.

In most fields of scientific endeavor, the outcomes of important experiments are not always known before the experiments are performed. But in protein structure prediction, algorithms are usually developed and tested in situations where the answers are known. In December 1996, the Second Meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2) was held in Asilomar, California to rectify this situation: protein sequences were provided in advance for which the experimental structure had not yet been published. Over 70 research groups provided bona fide predictions on 42 targets in four categories: comparative or 'homology' modeling, fold recognition or 'threading', ab initio structure predictions, and docking predictions. Since the previous CASP meeting in 1994, the role of fold recognition in structure prediction has increased enormously with the largest number of groups participating in this category. In this review, we highlight some of the important developments and give at least a qualitative sense of what kind of methods produced some of the better predictions.