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Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites.

ABSTRACT: The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 A, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca2+-binding and Na+-binding sites. The Ca2+ cation is located in domain 1 in a configuration very similar to that found in the activated bovine factor Va. The Na+ sites appear to play a structural role in providing rigidity to the three protuberances on the top surface of the molecule. These features probably help to steer substrates towards the mononuclear copper sites prior to their oxidation and to restrict the size of the approaching substrate. The trinuclear copper centre appears to differ from the room-temperature structure in that a dioxygen moiety is bound in a similar way to that found in the endospore coat protein CotA from Bacillus subtilis.

SUBMITTER: Bento I

PROVIDER: S-EPMC2483498 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites.

Bento Isabel I Peixoto Cristina C Zaitsev Vjacheslav N VN Lindley Peter F PF

Acta crystallographica. Section D, Biological crystallography 20070116 Pt 2

The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 A, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca2+-binding and Na+-binding sites. The Ca2+ cation is located in domain ...[more]

PMID: 17242517

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Project description:BackgroundUnderstanding the residue covariations between multiple positions in protein families is very crucial and can be helpful for designing protein engineering experiments. These simultaneous changes or residue coevolution allow protein to maintain its overall structural-functional integrity while enabling it to acquire specific functional modifications. Despite the significant efforts in the field there is still controversy in terms of the preferable locations of coevolved residues on different regions of protein molecules, the strength of coevolutionary signal and role of coevolution in functional diversification.MethodologyIn this paper we study the scale and nature of residue coevolution in maintaining the overall functionality and structural integrity of proteins. We employed a large scale study to investigate the structural and functional aspects of coevolved residues. We found that the networks representing the coevolutionary residue connections within our dataset are in general of 'small-world' type as they have clustering coefficient values higher than random networks and also show smaller mean shortest path lengths similar and/or lower than random and regular networks. We also found that altogether 11% of functionally important sites are coevolved with any other sites. Active sites are found more frequently to coevolve with any other sites (15%) compared to protein (11%) and ligand (9%) binding sites. Metal binding and active sites are also found to be more frequently coevolved with other metal binding and active sites, respectively. Analysis of the coupling between coevolutionary processes and the spatial distribution of coevolved sites reveals that a high fraction of coevolved sites are located close to each other. Moreover, approximately 80% of charge compensatory substitutions within coevolved sites are found at very close spatial proximity (<or= 5A), pointing to the possible preservation of salt bridges in evolution.ConclusionOur findings show that a noticeable fraction of functionally important sites undergo coevolution and also point towards compensatory substitutions as a probable coevolutionary mechanism within spatially proximal coevolved functional sites.

Project description:Biosynthesis of starch is catalyzed by a cascade of enzymes. The activity of a large number of these enzymes depends on interaction with polymeric substrates via carbohydrate binding sites, which are situated outside of the catalytic site and its immediate surroundings including the substrate-binding crevice. Such secondary binding sites can belong to distinct starch binding domains (SBDs), classified as carbohydrate binding modules (CBMs), or be surface binding sites (SBSs) exposed on the surface of catalytic domains. Currently in the Carbohydrate-Active enZYmes (CAZy) database SBDs are found in 13 CBM families. Four of these families; CBM20, CBM45, CBM48, and CBM53 are represented in enzymes involved in starch biosynthesis, namely starch synthases, branching enzymes, isoamylases, glucan, water dikinases, and α-glucan phosphatases. A critical role of the SBD in activity has not been demonstrated for any of these enzymes. Among the well-characterized SBDs important for starch biosynthesis are three CBM53s of Arabidopsis thaliana starch synthase III, which have modest affinity. SBSs, which are overall less widespread than SBDs, have been reported in some branching enzymes, isoamylases, synthases, phosphatases, and phosphorylases active in starch biosynthesis. SBSs appear to exert roles similar to CBMs. SBSs, however, have also been shown to modulate specificity for example by discriminating the length of chains transferred by branching enzymes. Notably, the difference in rate of occurrence between SBDs and SBSs may be due to lack of awareness of SBSs. Thus, SBSs as opposed to CBMs are not recognized at the protein sequence level, which hampers their identification. Moreover, only a few SBSs in enzymes involved in starch biosynthesis have been functionally characterized, typically by structure-guided site-directed mutagenesis. The glucan phosphatase Like SEX4 2 from A. thaliana has two SBSs with weak affinity for β-cyclodextrin, amylose and amylopectin, which were indicated by mutational analysis to be more important than the active site for initial substrate recognition. The present review provides an update on occurrence of functional SBDs and SBSs in enzymes involved in starch biosynthesis.

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Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites.

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Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites.

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