María Silvina Fornasari

Gamma carbonic anhydrases in plant mitochondria

Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (γCA), a Zn containing enzyme from Methanosarcina thermophila(CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of γCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from α and γ proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain γCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.

Gamma carbonic anhydrase like complex interact with plant mitochondrial complex I

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtγCAL1 and AtγCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtγCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtγCA and AtγCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtγCAs. All plant species analyzed contain both AtγCA and AtγCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtγCAL sequences show a deviation of functionally important active site residues with respect to γCAs but could form active interfaces in the interaction with AtγCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.

Functional and structural characterization of the catalytic domain of the starch synthase III from Arabidopsis thaliana.

Glycogen and starch are the major energy storage compounds in most living organisms. The metabolic pathways leading to their synthesis involve the action of several enzymes, among which glycogen synthase (GS) or starch synthase (SS) catalyze the elongation of the α‐1,4‐glucan backbone. At least five SS isoforms were described in Arabidopsis thaliana; it has been reported that the isoform III (SSIII) has a regulatory function on the synthesis of transient plant starch. The catalytic C‐terminal domain of A. thaliana SSIII (SSIII‐CD) was cloned and expressed. SSIII‐CD fully complements the production of glycogen by an Agrobacterium tumefaciens glycogen synthase null mutant, suggesting that this truncated isoform restores in vivo the novo synthesis of bacterial glycogen. In vitro studies revealed that recombinant SSIII‐CD uses with more efficiency rabbit muscle glycogen than amylopectin as primer and display a high apparent affinity for ADP‐Glc. Fold class assignment methods followed by homology modeling predict a high global similarity to A. tumefaciens GS showing a fully conservation of the ADP‐binding residues. On the other hand, this comparison revealed important divergences of the polysaccharide binding domain between AtGS and SSIII‐CD. Proteins 2008.

Starch‐synthase III family encodes a tandem of three starch‐binding domains

The starch‐synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N‐terminal transit peptide followed by a 557‐amino acid SSIII‐specific domain (SSIII‐SD) with three internal repeats and a C‐terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch‐binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved. Proteins 2006.

CoDNaS: a database of conformational diversity in the native state of proteins.

MOTIVATION Conformational diversity is a key concept in the understanding of different issues related with protein function such as the study of catalytic processes in enzymes, protein-protein recognition, protein evolution and the origins of new biological functions. Here, we present a database of proteins with different degrees of conformational diversity. Conformational Diversity of Native State (CoDNaS) is a redundant collection of three-dimensional structures for the same protein derived from protein data bank. Structures for the same protein obtained under different crystallographic conditions have been associated with snapshots of protein dynamism and consequently could characterize protein conformers. CoDNaS allows the user to explore global and local structural differences among conformers as a function of different parameters such as presence of ligand, post-translational modifications, changes in oligomeric states and differences in pH and temperature. Additionally, CoDNaS contains information about protein taxonomy and function, disorder level and structural classification offering useful information to explore the underlying mechanism of conformational diversity and its close relationship with protein function. Currently, CoDNaS has 122 122 structures integrating 12 684 entries, with an average of 9.63 conformers per protein. AVAILABILITY The database is freely available at http://www.codnas.com.ar/.

Protein Conformational Diversity Modulates Sequence Divergence

It is well established that the conservation of protein structure during evolution constrains sequence divergence. The conservation of certain physicochemical environments to preserve protein folds and then the biological function originates a site-specific structurally constrained substitution pattern. However, protein native structure is not unique. It is known that the native state is better described by an ensemble of conformers in a dynamic equilibrium. In this work, we studied the influence of conformational diversity in sequence divergence and protein evolution. For this purpose, we derived a set of 900 proteins with different degrees of conformational diversity from the PCDB database, a conformer database. With the aid of a structurally constrained protein evolutionary model, we explored the influence of the different conformations on sequence divergence. We found that the presence of conformational diversity strongly modulates the substitution pattern. Although the conformers share several of the structurally constrained sites, 30% of them are conformer specific. Also, we found that in 76% of the proteins studied, a single conformer outperforms the others in the prediction of sequence divergence. It is interesting to note that this conformer is usually the one that binds ligands participating in the biological function of the protein. The existence of a conformer-specific site-substitution pattern indicates that conformational diversity could play a central role in modulating protein evolution. Furthermore, our findings suggest that new evolutionary models and bioinformatics tools should be developed taking into account this substitution bias.

Protein Conformational Diversity Correlates with Evolutionary Rate

Native state of proteins is better represented by an ensemble of conformers in equilibrium than by only one structure. The extension of structural differences between conformers characterizes the conformational diversity of the protein. In this study, we found a negative correlation between conformational diversity and protein evolutionary rate. Conformational diversity was expressed as the maximum root mean square deviation (RMSD) between the available conformers in Conformational Diversity of Native State database. Evolutionary rate estimations were calculated using 16 different species compared with human sharing at least 700 orthologous proteins with known conformational diversity extension. The negative correlation found is independent of the protein expression level and comparable in magnitude and sign with the correlation between gene expression level and evolutionary rate. Our findings suggest that the structural constraints underlying protein dynamism, essential for protein function, could modulate protein divergence.

On the effect of protein conformation diversity in discriminating among neutral and disease related single amino acid substitutions

BackgroundNon-synonymous coding SNPs (nsSNPs) that are associated to disease can also be related with alterations in protein stability. Computational methods are available to predict the effect of single amino acid substitutions (SASs) on protein stability based on a single folded structure. However, the native state of a protein is not unique and it is better represented by the ensemble of its conformers in dynamic equilibrium. The maintenance of the ensemble is essential for protein function. In this work we investigated how protein conformational diversity can affect the discrimination of neutral and disease related SASs based on protein stability estimations. For this purpose, we used 119 proteins with 803 associated SASs, 60% of which are disease related. Each protein was associated with its corresponding set of available conformers as found in the Protein Conformational Database (PCDB). Our dataset contains proteins with different extensions of conformational diversity summing up a total number of 1023 conformers.ResultsThe existence of different conformers for a given protein introduces great variability in the estimation of the protein stability (ΔΔG) after a single amino acid substitution (SAS) as computed with FoldX. Indeed, in 35% of our protein set at least one SAS can be described as stabilizing, destabilizing or neutral when a cutoff value of ±2 kcal/mol is adopted for discriminating neutral from perturbing SASs. However, when the ΔΔG variability among conformers is taken into account, the correlation among the perturbation of protein stability and the corresponding disease or neutral phenotype increases as compared with the same analysis on single protein structures. At the conformer level, we also found that the different conformers correlate in a different way to the corresponding phenotype.ConclusionsOur results suggest that the consideration of conformational diversity can improve the discrimination of neutral and disease related protein SASs based on the evaluation of the corresponding Gibbs free energy change.

CoDNaS 2.0: a comprehensive database of protein conformational diversity in the native state.

CoDNaS (conformational diversity of the native state) is a protein conformational diversity database. Conformational diversity describes structural differences between conformers that define the native state of proteins. It is a key concept to understand protein function and biological processes related to protein functions. CoDNaS offers a well curated database that is experimentally driven, thoroughly linked, and annotated. CoDNaS facilitates the extraction of key information on small structural differences based on protein movements. CoDNaS enables users to easily relate the degree of conformational diversity with physical, chemical and biological properties derived from experiments on protein structure and biological characteristics. The new version of CoDNaS includes ∼70% of all available protein structures, and new tools have been added that run sequence searches, display structural flexibility profiles and allow users to browse the database for different structural classes. These tools facilitate the exploration of protein conformational diversity and its role in protein function. Database URL: http://ufq.unq.edu.ar/codnas

Dynactins p25 and p27 are predicted to adopt the LβH fold

Dynactin is a multimeric protein essential for the minus‐end‐directed transport driven by microtubule‐based motor dynein. The pointed‐end subcomplex in dynactin contains p62, p27, p25, and Arp11 subunits, and is thought to participate in interactions with membranous cargoes. We used sequence and structure prediction analysis to study dynactins p25 and p27. Here we present evidence that strongly supports that dynactins p27 and p25 contain the isoleucine‐patch motif and adopt the left‐handed parallel β‐helix fold. The structural models we obtained could contribute to the understanding of the complex interactions that dynactins are able to establish with cargo particles, microtubules or other dynactin subunits.