In-silico Assessment of Protein-Protein Electron Transfer. A Case Study: Cytochrome c Peroxidase – Cytochrome c

<div><p>The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, <i>ab-initio</i> quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET) processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT) through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47×10⁶ s⁻¹ for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191.</p> </div

Electron transfer region of the CcP/Cytc complex.

<p>The ET pathway proposed by Pelletier and Kraut is shown in red, the ET pathway suggested by Siddarth is shown in blue.</p

Average distances <i>d</i> in Å, Electronic coupling <i>rmsV</i> in eV, Δ<i>G</i>° in eV, λ in eV and in s⁻¹ calculated for HT between donor and acceptor (DA), donor and bridge (DB), and bridge and acceptor (BA), respectively.

<p>The electronic coupling is calculated applying QM setups <i>direct</i>, <i>full</i>, <i>path1</i> and <i>path2</i>. <i>k_ET</i> is calculated by Marcus theory applying the respective highest electronic coupling of the system. Fluctuations are depicted through the coherence factor given in parentheses.</p

Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of &gt;5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme-ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e(-70)) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally

Intermediates and products of Channel C.

<p>Intermediates and products of Channel C.</p

Illustration of the key dihedral angle C16-C17-C18-H18 that determines the conversion of I1 to I2: a) A-I1; b) B-I1.

<p>Illustration of the key dihedral angle C16-C17-C18-H18 that determines the conversion of I1 to I2: a) A-I1; b) B-I1.</p

Example reactions of TPSs: a) limonene synthase; b) squalene-hopene cyclase.

<p>Example reactions of TPSs: a) limonene synthase; b) squalene-hopene cyclase.</p

A hypothetical example output of the carbocation docking.

<p>A hypothetical example output of the carbocation docking.</p

Docking score (MM/GBSA) of 9 carbocationic intermediates for 22 triterpenoid synthase homology models that follow channel C.

<p>Compounds that could not be successfully docked at all are arbitrarily assigned a docking score of −10 kcal/mol. Figure legend shows the UniProtKB IDs for the triterpenoid synthases. Panel a shows the docking scores against 8 lanosterol synthases (in red); panel b shows the docking scores against 10 cycloartenol synthases (in lime green); and panel c shows the docking scores against a cucurbitadienol synthase (in cyan), a parkeol synthase (in magenta) and 2 protostadienol synthases (in blue). Details c.f. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003874#pcbi.1003874.s006" target="_blank">Table S2</a>.</p