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Results for “In silico modeling”

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PubMed2006

Polarization and movement of keratocytes: a multiscale modelling approach.

Eukariotic cell motility is a complex phenomenon, in which the cytoskeleton and its major constituent, actin, play an essential role. Actin forms polymers of long, stiff filaments that are cross-linked into an anisotropic network inside a thin sheet-like cellular protrusion, the lamellipod. At the leading edge of this structure, polymerization of actin filaments creates the force that pushes out the membrane and leads to translocation of a motile cell. Dynamics of the actin network account for changes in cell shape, crawling motion and turning of the cell in response to external cues. Regulating the dynamics of the cytoskeleton, and playing a central role in signal transduction in the cell, are Cdc42, Rac and Rho (GTPases of the rho family, collectively known as the small G-proteins) and the actin nucleating complex, Arp2/3. In this paper, we use a multiscale modelling approach in a 2D model of a motile cell. We describe the mutual interactions of the small G-proteins, and their effects on capping and side-branching of actin filaments. We incorporate the pushing exerted by oriented actin filament ends on the cell edge, and a Rho-dependent contraction force. Combining these biochemical and mechanical aspects, we investigate the dynamics of a model epidermal fish keratocyte through in silico experiments. Our model gives insight into how, in response to some cue, a cell can polarize, form a leading edge, and move; concomitantly it explains how a keratocyte cell can maintain its shape and polarity, even after removal of the initial stimulus, and how it can change direction quickly in response to changes in its environment. We show that establishment of polarity stems from interactions of Cdc42, Rac and Rho, while maintenance and robustness of polarity is due to the rapid cytosolic diffusion of the inactive (GDI-bound) forms of the small G-proteins. Our model produces a cell shape that closely resembles the keratocytes and correct speeds for biologically reasonable parameter values. Movies of the simulations can be obtained from http://theory.bio.uu.nl/stan/keratocyte.

Actins↗

PubMed2006

Phosphorylation of Thr654 but not Thr669 within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding.

Calcium-calmodulin (CaM) binding to the epidermal growth factor receptor (EGFR) has been shown to both inhibit and stimulate receptor activity. CaM binds to the intracellular juxtamembrane (JM) domain (Met645-Phe688) of EGFR. Protein kinase C (PKC) mediated phosphorylation of Thr654 occurs within this domain. CaM binding to the JM domain inhibits PKC phosphorylation and conversely PKC mediated phosphorylation of Thr654 or Glu substitution of Thr654 inhibits CaM binding. A second threonine residue (Thr669) within the JM domain is phosphorylated by the mitogen-activated protein kinase (MAPK). Previous results have shown that CaM interferes with EGFR-induced MAPK activation. If and how phosphorylation of Thr669 affects CaM-EGFR interaction is however not known. In the present study we have used surface plasmon resonance (BIAcore) to study the influence of Thr669 phosphorylation on real time interactions between the intracellular juxtamembrane (JM) domain of EGFR and CaM. The EGFR-JM was expressed as GST fusion proteins in Escherichia coli and phosphorylation was mimicked by generating Glu substitutions of either Thr654 or Thr669. Purified proteins were coupled to immobilized anti-GST antibodies at the sensor surface and increasing concentration of CaM was applied. When mutating Thr654 to Glu654 no specific CaM binding could be detected. However, neither single substitutions of Thr669 (Gly669 or Glu669) nor double mutants Gly654/Gly669 or Gly654/Glu669 influenced the binding of CaM to the EGFR-JM. This clearly shows that PKC may regulate EGF-mediated CaM signalling through phosphorylation of Thr654 whereas phosphorylation of Thr669 seems to play a CaM independent regulatory role. The role of both residues in the EGFR-calmodulin interaction was also studied in silico. Our modelling work supports a scenario where Thr654 from the JM domain interacts with Glu120 in the calmodulin molecule. Phosphorylation of Thr654 or Glu654 substitution creates a repulsive electrostatic force that would diminish CaM binding to the JM domain. These results are in line with the Biacore experiments showing a weak binding of the CaM to the JM domain with Thr654 mutated to Glu. Furthermore, these results provide a hypothesis to how CaM binding to EGFR might both positively and negatively interfere with EGFR-activity.

Binding Sites↗

PubMed2005

Recognizing and defining true Ras binding domains II: in silico prediction based on homology modelling and energy calculations.

Considering the large number of putative Ras effector proteins, it is highly desirable to develop computational methods to be able to identify true Ras binding molecules. Based on a limited sequence homology among members of the Ras association (RA) and Ras binding (RB) sub-domain families of the ubiquitin super-family, we have built structural homology models of Ras proteins in complex with different RA and RB domains, using the FOLD-X software. A critical step in our approach is to use different templates of Ras complexes, in order to account for the structural variation among the RA and RB domains. The homology models are validated by predicting the effect of mutating hot spot residues in the interface, and residues important for the specificity of interaction with different Ras proteins. The FOLD-X calculated energies of the best-modelled complexes are in good agreement with previously published experimental data and with new data reported here. Based on these results, we can establish energy thresholds above, or below which, we can predict with 96% confidence that a RA/RB domain will or will not interact with Ras. This study shows the importance of in depth structural analysis, high quality force-fields and modelling for correct prediction. Our work opens the possibility of genome-wide prediction for this protein family and for others, where there is enough structural information.

Amino Acid Sequence↗

PubMed2004

New predictors for several ADME/Tox properties: aqueous solubility, human oral absorption, and Ames genotoxicity using topological descriptors.

In silico predictive models for aqueous solubility, human intestinal absorption (HIA), and Ames genotoxicity were developed principally using artificial neural net (ANN) analysis and topological descriptors. Approximately 10,000 compounds spread across three data sets were used in the construction of these quantitative-structure-activity/property-relationship (QSAR/QSPR) models. For aqueous solubility, 5,037 chemically diverse compounds were used to construct ANN-QSPRs for intrinsic aqueous solubility. When these robust models were applied to 938 compounds in external validation, they gave an r2 = 0.78 with 84% predicted within 1 log unit for these new chemical entities (NCEs). 417 therapeutic drugs were used in the development of an ANN-QSPR to predict for percent oral absorption (%OA). For validation testing on 195 new drugs, 92% of the compounds were predicted to within 25% of their reported %OA values, which ranged from 0% to 100%. Polar surface area and logP, the octanol-water partition coefficient, were found to be important descriptors in our QSPR model. Development of an ANN-QSAR as a genotoxicity predictor for S. typhimurium employed 2963 compounds including 290 therapeutic drugs. Validation results on 400 NCEs with the ANN-QSAR gave a concordance of 83% which rose to 91% when a confidence indicator was applied. With new drugs a concordance of 92% was reached, which increased to 97% when the reliably indicator was invoked.

Administration, Oral↗

PubMed2004

Metabolic network analysis of the causes and evolution of enzyme dispensability in yeast.

Under laboratory conditions 80% of yeast genes seem not to be essential for viability. This raises the question of what the mechanistic basis for dispensability is, and whether it is the result of selection for buffering or an incidental side product. Here we analyse these issues using an in silico flux model of the yeast metabolic network. The model correctly predicts the knockout fitness effects in 88% of the genes studied and in vivo fluxes. Dispensable genes might be important, but under conditions not yet examined in the laboratory. Our model indicates that this is the dominant explanation for apparent dispensability, accounting for 37-68% of dispensable genes, whereas 15-28% of them are compensated by a duplicate, and only 4-17% are buffered by metabolic network flux reorganization. For over one-half of those not important under nutrient-rich conditions, we can predict conditions when they will be important. As expected, such condition-specific genes have a more restricted phylogenetic distribution. Gene duplicates catalysing the same reaction are not more common for indispensable reactions, suggesting that the reason for their retention is not to provide compensation. Instead their presence is better explained by selection for high enzymatic flux.

Biomass↗

PubMed2007

Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli.

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of beta-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

Amidohydrolases↗

PubMed2003

The anomalous pKa of Tyr-9 in glutathione S-transferase A1-1 catalyzes product release.

The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.

Binding Sites↗

PubMed2003

Optimizing the search algorithm for protein engineering by directed evolution.

An in silico protein model based on the Kauffman NK-landscape, where N is the number of variable positions in a protein and K is the degree of coupling between variable positions, was used to compare alternative search strategies for directed evolution. A simple genetic algorithm (GA) was used to model the performance of a standard DNA shuffling protocol. The search effectiveness of the GA was compared to that of a statistical approach called the protein sequence activity relationship (ProSAR) algorithm, which consists of two steps: model building and library design. A number of parameters were investigated and found to be important for the comparison, including the value of K, the screening size, the system noise and the number of replicates. The statistical model was found to accurately predict the measured activities for small values of the coupling between amino acids, K <or= 1. The ProSAR strategy was found to perform well for small to moderate values of coupling, 0 <or= K <or= 3, and was found to be robust to noise. Some implications for protein engineering are discussed.

Algorithms↗

PubMed2005

Meeting report: the future and limits of systems biology.

Systems biology has enjoyed explosive growth in both the number of people participating in this area of research and the number of publications on the topic. And yet, the paradigms that underlie the field have not seen a similar expansiveness. Instead, most of these paradigms have been carried over from other fields like engineering, physics, and mathematics. As a result, a small set of concepts dominate the field. The traditional biologist is seen by many as outmoded and tolerated only as a source of data. In this view, the biologist's ideas may even be considered conceptually and theoretically irrelevant. In this Perspective, we take a critical look at some of the paradigms of systems biology and question whether the biologist's ideas, methods, and theories have really become outmoded. We see the future of systems biology as a tight coupling of in vivo and in vitro methods for bioengineering with in silico multicellular modeling and simulation.

Computational Biology↗

PubMed2026

De Novo TRIO Missense Variants Disrupt Ras-GEF Domains and Cause Congenital Ventriculomegaly and Hydrocephalus.

Congenital hydrocephalus (CH), characterized by congenital ventriculomegaly (CV), affects approximately 0.5-1 per 1000 live births and is a common cause of pediatric neurosurgical intervention, yet its genetic architecture remains incompletely defined. We report a child with syndromic CH requiring cerebrospinal fluid diversion who harbored a pathogenic de novo missense variant in TRIO (c.3232C > T; p.(Arg1078Trp)), a gene previously associated with autosomal dominant neurodevelopmental disorders featuring variable head circumference. This case prompted systematic evaluation of TRIO variation in our CV/CH cohort (2,697 patient-parent trios) using exome sequencing. We identified five additional unrelated probands with de novo TRIO variants, including two novel substitutions affecting the same residue within the Ras-GEF1 domain (p.(Glu1299Lys) and p.(Glu1299Gly)), yielding significant gene-level enrichment for protein-damaging de novo variants (adjusted p = 6.12 × 10-5). All affected individuals exhibited CV, frequently accompanied by developmental delay and additional structural brain abnormalities. In silico structural modeling predicted that associated variants destabilize critical TRIO Ras-GEF domains required for Rho GTPase activation. Analysis of single-nucleus transcriptomic data from the developing human neocortex revealed enrichment of TRIO expression in multipotent progenitor populations. A systematic literature review identified six additional individuals with TRIO de novo variants and reported CV or CH, including an unrelated patient with the same p.(Arg1078Trp) substitution. Together, these findings expand the phenotypic spectrum associated with pathogenic TRIO variation to include CV/CH and support TRIO as a clinically relevant gene in the genetic evaluation of syndromic CV/CH patients.

Humans↗

PubMed2005

Mast cell-specific genes--new drug targets/pathogenesis.

It has become possible to see all the expressed genes present in a cell (transcriptome) at once using microarray. We have applied microarray technology in various studies involving allergic diseases. Although we and others have discovered various novel molecules crucially involved in the pathogenesis of the disease, transcriptome assay is now expected as a tool for understanding the whole molecule balancing, i.e., system biology. Here I introduce examples of our trials for understanding the whole functional roles of mast cells as has been published in the web database for transcriptomes expressed by several mast cell types and various cell types. In the near future, we will be able to construct human mast cell models in silico (in a computer) by analyzing integrative information regarding the genome, transcriptome and proteome of mast cells, and will be able to test our hypotheses without having to perform in vitro tests.

Animals↗

PubMed2005

Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene.

PURPOSE: Pigmented paravenous chorioretinal atrophy (PPCRA) is an unusual retinal degeneration characterized by accumulation of pigmentation along retinal veins. The purpose of this study was to describe the phenotype of a family with PPCRA, determine the mode of inheritance, and identify the causal mutation. METHODS: Ophthalmic examination was performed on seven family members and serially detailed in the proband over a 3-year period. Blood samples were collected and DNA extracted. All 12 coding exons and the 5' promoter region of the crumbs homologue 1 (CRB1) gene were PCR amplified and DNA sequenced. In silico homology modeling was performed on the mutated protein domain. RESULTS: Subtle symmetrical chorioretinal atrophy in the inferior quadrant was the earliest clinical sign detectable within this family. Paravenous pigmentation occurred initially in the far periphery, progressing centrally, with atrophy later becoming more widespread, involving the nasal, then the temporal, and finally the upper quadrant. A novel, dominant Val162Met mutation within the fourth EGF-like domain of CRB1 cosegregates with the PPCRA phenotype. It is thought to affect domain structure, because codon 162 is involved in hydrogen bonding between the antiparallel beta-strands of the major beta-sheet, causing sufficient perturbation of the backbone that the domain-stabilizing hydrogen bond does not form or is weakened. CONCLUSIONS: PPCRA was dominantly inherited in this family, but exhibited variable expressivity. Males are more likely to exhibit a severe phenotype, whereas females may remain virtually asymptomatic even in later years. The PPCRA phenotype is associated with a Val162Met mutation in CRB1 which is likely to affect the structure of the CRB1 protein.

Adult↗

PubMed2003

Two homozygous mutations in the 11 beta-hydroxysteroid dehydrogenase type 2 gene in a case of apparent mineralocorticoid excess.

The human microsomal 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) metabolizes active cortisol into cortisone and protects the mineralocorticoid receptor from glucocorticoid occupancy. In a congenital deficiency of 11 beta-HSD2, the protective mechanism fails and cortisol gains inappropriate access to mineralocorticoid receptor, resulting in low-renin hypertension and hypokalemia. In the present study, we describe the clinical and molecular genetic characterization of a patient with a new mutation in the HSD11B2 gene. This is a 4-yr-old male with arterial hypertension. The plasma renin activity and serum aldosterone were undetectable in the presence of a high cortisol to cortisone ratio. PCR amplification and sequence analysis of HSD11B2 gene showed the homozygous mutation in exon 4 Asp223Asn (GAC-->AAC) and a single nucleotide substitution C-->T in intron 3. Using site-directed mutagenesis, we generated a mutant 11 beta HSD2 cDNA containing the Asp223Asn mutation. Wild-type and mutant cDNA was transfected into Chinese hamster ovary cells and enzymatic activities were measured using radiolabeled cortisol and thin-layer chromatography. The mRNA and 11 beta HSD2 protein were detected by RT-PCR and Western blot, respectively. Wild-type and mutant 11 beta HSD2 protein was expressed in Chinese hamster ovary cells, but the mutant enzyme had only 6% of wild-type activity. In silico 3D modeling showed that Asp223Asn changed the enzyme's surface electrostatic potential affecting the cofactor and substrate enzyme-binding capacity. The single substitution C-->T in intron 3 (IVS3 + 14 C-->T) have been previously reported that alters the normal splicing of pre-mRNA, given a nonfunctional protein. These findings may determine the full inactivation of this enzyme, explaining the biochemical profile and the early onset of hypertension seen in this patient.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2026

Host hybridization enabled the emergence of a reassorted hantavirus lineage.

The exchange of genetic material between individuals is a key driver of evolution and diversification across most branches of life. Segmented viruses can exchange genetic material through reassortment of genomic segments. New viral strains that emerge from reassortments can have greater infection ranges and higher virulence, although concrete examples of the adaptive advantages of reassortants in nature apart from influenza remain rare. We studied here the evolutionary history and consequences of reassortment in Tula orthohantavirus (TULV) in a hybrid zone between evolutionary lineages of its reservoir host, the common vole (Microtus arvalis). Across 58 trapping sites and 127 infected voles, we detected 27 TULV reassortants in a 12.5 km broad zone at the contact of the parental TULV clades, resembling a viral hybrid zone concordant with the hosts'. Phylogenomic analyses revealed three independent reassortment events, but most of the host hybrid zone was dominated by a single strain with a reassorted M-Segment, which encodes the surface glycoprotein. We detected clade-specific variation in the glycoprotein's N-terminal region consisting of five residues, two of which showed evidence of positive selection. In silico 3D modeling of seven glycoproteins confirmed that this N-terminal region has a unique and specific structure for each TULV clade and the dominant reassortants and is the only structurally variable region of the TULV glycoprotein. Our findings suggest that reassortment between the parental TULV clades in the contact region has resulted in a transgressive virus phenotype potentially adapted to hybrid hosts. This demonstrates the potential of zones of hybridization for the emergence of new virus strains with novel evolutionary trajectories.

Animals↗

PubMed2005

Predictive ADME-Tox London, UK, 27 -- 28 April 2005.

The Predictive ADME-Tox conference brought together scientists responsible for innovative in vitro and in silico screens to predict particular chemical liabilities. Oral bioavailability without adverse effects requires good absorption, distribution, metabolism and excretion (ADME) and minimal toxicity. Traditionally, these properties are measured late in development, when sufficient material is available for classical assays. Although these assays remain the gold standard, the conference focused on novel methods to predict liabilities early in the development pipeline, when a course change has less impact in the development timeline. Emerging themes from the meeting were i) integration - of data from a number of predictive assays into a 'profile' of a new class, and of safety scientists into discovery teams, in order to guide interpretation of the profile and identify tests to include in the hit-to-lead process; and ii) collaboration. As in silico predictive models are increasingly accepted, from reactivity and genetic toxicity to models for hERG inhibition, kinase crossreactivity and cytochrome P450 reactivity, increasing amounts of high-quality data will be needed to 'feed' the models. Such data will soon be beyond the capabilities of individual organisations to generate, and methods for pooling data are needed to advance the generation of predictive models for new toxicities. In addition, collaboration to apply novel methods such as metabonomics profiling to withdrawn drugs may permit the identification of new safety biomarkers to prevent such costly failures.

Biotransformation↗

PubMed2006

A computational protocol for the integration of the monotopic protein prostaglandin H2 synthase into a phospholipid bilayer.

Prostaglandin H2 synthase (PGHS) synthesizes PGH2, a prostaglandin precursor, from arachidonic acid and was the first monotopic enzyme to have its structure experimentally determined. Both isozymes of PGHS are inhibited by nonsteroidal antiinflammatory drugs, an important class of drugs that are the primary means of relieving pain and inflammation. Selectively inhibiting the second isozyme, PGHS-2, minimizes the gastrointestinal side-effects. This had been achieved by the new PGHS-2 selective NSAIDs (i.e., COX-2 inhibitors) but it has been recently suggested that they suffer from additional side-effects. The design of these drugs only made use of static structures from x-ray crystallographic experiments. Investigating the dynamics of both PGHS-1 and PGHS-2 using classical molecular dynamics is expected to generate new insight into the differences in behavior between the isozymes, and therefore may allow improved PGHS-2 selective inhibitors to be designed. We describe a molecular dynamics protocol that integrates PGHS monomers into phospholipid bilayers, thereby producing in silico atomistic models of the PGHS system. Our protocol exploits the vacuum created beneath the protein when several lipids are removed from the top leaflet of the bilayer. The protein integrates into the bilayer during the first 5 ns in a repeatable process. The integrated PGHS monomer is stable and forms multiple hydrogen bonds between the phosphate groups of the lipids and conserved basic residues (Arg, Lys) on the protein. These interactions stabilize the system and are similar to interactions observed for transmembrane proteins.

Algorithms↗

PubMed2006

Inflamm-aging, cytokines and aging: state of the art, new hypotheses on the role of mitochondria and new perspectives from systems biology.

In this article we summarise present knowledge on the role of pro-inflammatory cytokines on chronic inflammation leading to organismal aging, a phenomenon we proposed to call "inflamm-aging". In particular, we review genetic data regarding polymorphisms of genes encoding for cytokines and proteins involved in natural immunity (such as Toll-like Receptors and Heat Shock Proteins) obtained from large population studies including young, old and very old people in good health status or affected by age-related diseases such as Alzheimer's Disease and Type II Diabetes. On the whole, despite some controversial results, the available data are in favour of the hypothesis that pro-inflammatory cytokines play an important role in aging and longevity. Further, we present a possible hypothesis to reconcile energetic dysfunction, including mitochondria, and inflamm-aging. New perspectives for future studies, including phylogenetic studies in animal models and in silico studies on mathematical and bioinformatic models inspired by the systems biology approach, are also proposed.

Aging↗

PubMed2006

Evolution of a "conserved" amino acid sequence: a model study of an in silico investigation of the phylogenesis of some immune receptors.

In this paper we analyze a 55-amino acid (aa) sequence which is relatively well conserved in several seven-transmembrane receptor families (from Insects to Mammals) and in some Viruses. This sequence, which covers the second transmembrane domain, the first extracellular loop and the third transmembrane domain, appears in its complete configuration in most of the seven-transmembrane receptor families, as well as in the protein products of some viruses. Other seven-transmembrane receptors and viruses exhibit reduced configurations of the conserved sequence, lacking either aa 31 or aa 30-31. 53-aa configurations are typically found in most chemokine receptor (CKR) subfamilies, as well as in some viral protein products. However, the CCR1, CCR3, and CCR6 subfamilies comprise a 54-aa configuration and the CKR-related protein products, ChemR23 and RDC1, include the complete 55-aa sequence. For each CKR subfamily the "modal sequence" of the conserved segment was constructed by selecting the most frequently occurring aa at each position. Then, pairwise alignments were made between: (i) the modal CKR sequences, and (ii) the sequence (53-aa) of the Yaba-like disease virus - 7L protein. From the alignments two consensus matrices were derived: (i) the consensus 1 matrix with reference to the whole conserved segment, and (ii) the consensus 2 matrix with reference to aa 22-29, which appear to be the most variable segment of the sequence. Based on the obtained consensus values and with reference to this specific conserved segment, the following conclusions are proposed: (1) ChemR23 and RDC1 are probably the more primitive CKR forms; (2) CCR1 and CCR3 may be grouped in a single cluster; (3) CCRs 2, 4, and 5 are closely related to each other and may be grouped in a cluster; CCR7 is likely to be evolutionarily related to this cluster; (4) CXCRs 2, 3, and 4 and CCX CKR appear to be evolutionarily related to each other and very likely derived from an CCR6-like gene; (5) CCR2/4/5 and CCR7 may have derived either from CCR1/3-like or CCR6-like genes; (6). The Yaba-like disease virus--7L protein most likely derived, through "molecular piracy", from a CCR8-like gene. We also discuss possible, more remote, evolutionary links between CKRs, formylpeptide receptors, and possibly the highly conserved 18S rRNA genes.

Amino Acid Sequence↗