Energetic Selection of Topology in Ferredoxins

Models of early protein evolution posit the existence of short peptides that bound metals and ions and served as transporters, membranes or catalysts. The Cys-X-X-Cys-X-X-Cys heptapeptide located within bacterial ferredoxins, enclosing an Fe4S4 metal center, is an attractive candidate for such an early peptide. Ferredoxins are ancient proteins and the simple α+β fold is found alone or as a domain in larger proteins throughout all three kingdoms of life. Previous analyses of the heptapeptide conformation in experimentally determined ferredoxin structures revealed a pervasive right-handed topology, despite the fact that the Fe4S4 cluster is achiral. Conformational enumeration of a model CGGCGGC heptapeptide bound to a cubane iron-sulfur cluster indicates both left-handed and right-handed folds could exist and have comparable stabilities. However, only the natural ferredoxin topology provides a significant network of backbone-to-cluster hydrogen bonds that would stabilize the metal-peptide complex. The optimal peptide configuration (alternating αL,αR) is that of an α-sheet, providing an additional mechanism where oligomerization could stabilize the peptide and facilitate iron-sulfur cluster binding

Report Cloning and Characterization of Four Novel Coral Acid-Rich Proteins that Precipitate Carbonates In Vitro

Summary Biomineralization is a widely dispersed and highly regulated but poorly understood process by which organisms precipitate minerals from a wide variety of elements Results and Discussion Despite the broad interest in coral calcification and the potential for climate-driven adverse effects, the molecules and biophysical mechanism responsible for the precipitation of carbonates in corals are poorly understood. To date, we lack both a characterization of molecules involved in calcification and a mechanistic understanding of processes that lead to and control calcification. This lack of knowledge limits our ability to predict the response of corals to increasing atmospheric CO 2 . To date, the best-characterized highly acidic proteins that catalyze the precipitation of carbonates are from mollusk shells and an echinoder

Cloning and Characterization of Four Novel Coral Acid-Rich Proteins that Precipitate Carbonates In Vitro

SummaryBiomineralization is a widely dispersed and highly regulated but poorly understood process by which organisms precipitate minerals from a wide variety of elements [1]. For many years, it has been hypothesized that the biological precipitation of carbonates is catalyzed by and organized on an extracellular organic matrix containing a suite of proteins, lipids, and polysaccharides [2, 3]. The structures of these molecules, their evolutionary history, and the biophysical mechanisms responsible for calcification remain enigmatic. Despite the recognition that mineralized tissues contain proteins that are unusually rich in aspartic and glutamic acids [4–6], the role of these proteins in biomineralization remains elusive [5, 6]. Here we report, for the first time, the identification, cloning, amino acid sequence, and characterization of four highly acidic proteins, derived from expression of genes obtained from the common stony coral, Stylophora pistillata. Each of these four proteins can spontaneously catalyze the precipitation of calcium carbonate in vitro. Our results demonstrate that coral acid-rich proteins (CARPs) not only bind Ca2+ stoichiometrically but also precipitate aragonite in vitro in seawater at pH 8.2 and 7.6, via an electrostatic interaction with protons on bicarbonate anions. Phylogenetic analysis suggests that at least one of the CARPs arose from a gene fusion. Similar, highly acidic proteins appear to have evolved several times independently in metazoans through convergence. Based purely on thermodynamic grounds, the predicted change in surface ocean pH in the next decades would appear to have minimal effect on the capacity of these acid-rich proteins to precipitate carbonates

Topology angle in a ferredoxin fold for database analysis.

<p>An arbitrary plane was defined with three cysteine carbon alpha coordinates. Three dimensional vector calculations were done to determine the topology angle of the protein fold.</p

Fold topology in a ferredoxin fold.

<p>Right/Left fold configuration can be defined with an outlier, by orienting the outlier cysteine along the z-axis and iron-sulfur cluster being at the origin. A ferredoxin fold, with a conserved sequence CxxCxxC with an outlier cysteine, can create either right or left topological configuration. Right-handed fold is shown.</p

Cys-Gly-Gly-Clu-Gly-Gly-Cys peptide created with protCAD.

<p>All possible structures are explored by permuting 17 rotatable dihedral angles of the peptide from −180 to 180 with a step size of 60 degrees.</p

Protein ensemble generated by modifying psi, phi and chi dihedral angles.

<p>For a model heptapeptide-cluster complex, CGGCGGC fused to an iron-sulfur cluster, there are total 6 ψ angles, 6 Φ angles, 3 χ₁ angles, and one each for χ₂ and χ₃ angles. The permutations are carried out by 60 degrees step size for Φ and ψ and 120 degrees step size for χ angles.</p

The energy distributions of right (blue) and left-handed (red) structures.

<p>The gaussian fits are very similar, which suggests that the natural selection was not influenced by the energetic stability alone. The energy corresponding to the ensemble that has the lowest RMSD to the experimentally determined ferredoxin structure (PDB: 2FDN)- green circle.</p

Topology angles of entactic structural states.

<p>Cys-Gly-Gly-Clu-Gly-Gly-Cys hepeptide model has 232 structural entactic states, either right-handed (blue, 75 out of 232) or left-handed (red, 157 out of 232). Despite the inexistence of left-handed topological state in nature, model peptide suggests that left-handed structure can also properly interact with an iron-sulfur cluster.</p