doi:10.1016/j.jmb.2010.09.029 J. Mol. Biol. (2010) 404, 158–171 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb Coevolution Predicts Direct Interactions between mtDNA-Encoded and nDNA-Encoded Subunits of Oxidative Phosphorylation Complex I Moran Gershoni1†, Angelika Fuchs2†, Naama Shani1, Yearit Fridman1, Marisol Corral-Debrinski3, Amir Aharoni1, Dmitrij Frishman2⁎ and Dan Mishmar1⁎ 1Department of Life Sciences and the Nation Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel 2Technische Universität München, Wissenschaftszentrum Weihenstephan, Am Forum 1, 85354 Freising, Germany 3Institut de la Vision, Université Pierre et Marie Curie Paris 6, Unité Mixte de Recherche, S 592, 17 rue Moreau, Paris F-75012, France Received 29 October 2009; Despite years of research, the structure of the largest mammalian received in revised form oxidative phosphorylation (OXPHOS) complex, NADH–ubiquinone oxi- 5 September 2010; doreductase (complex I), and the interactions among its 45 subunits are accepted 13 September 2010 not fully understood. Since complex I harbors subunits encoded by Available online mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) genomes, with 22 September 2010 the former evolving ∼10 times faster than the latter, tight cytonuclear coevolution is expected and observed. Recently, we identified three Edited by M. Sternberg nDNA-encoded complex I subunits that underwent accelerated amino acid replacement, suggesting their adjustment to the elevated mtDNA rate Keywords: of change. Hence, they constitute excellent candidates for binding coevolution; mtDNA-encoded subunits. complex I; Here, we further disentangle the network of physical cytonuclear correlated mutation analysis; interactions within complex I by analyzing subunits coevolution. Firstly, mitochondrial DNA; relying on the bioinformatic analysis of 10 protein complexes possessing mitochondria solved structures, we show that signals of coevolution identified physically interacting subunits with nearly 90% accuracy, thus lending support to our approach. When applying this approach to cytonuclear interaction within complex I, we predict that the ‘rate-accelerated’ nDNA-encoded subunits of complex I, NDUFC2 and NDUFA1, likely interact with the mtDNA-encoded subunits ND5/ND4 and ND5/ND4/ND1, respectively. Furthermore, we predicted interactions among mtDNA-encoded complex I subunits. Using the yeast two-hybrid system, we experimentally confirmed the predicted interactions of human NDUFC2 with ND4, the interactions of human NDUFA1 with ND1 and ND4, and the lack of interaction of NDUFC2 with ND3 and NDUFA1, thus providing a proof of concept for our approach. *Corresponding authors. E-mail addresses: [email protected]; [email protected]. † M.G. and A.F. contributed equally to this study. Abbreviations used: OXPHOS, oxidative phosphorylation; complex I, NADH–ubiquinone oxidoreductase; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; McBASC, McLachlan-based substitution correlation; OMES, observed minus expected squared; ELSC, explicit likelihood of subset covariation; ROC, receiver operator characteristic; AUC, area under the curve. 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved. Coevolution in Oxidative Phosphorylation Complex I 159 Our study shows, for the first time, evidence for direct interactions between nDNA-encoded and mtDNA-encoded subunits of human OXPHOS complex I and paves the path towards deciphering subunit interactions within complexes lacking three-dimensional structures. Our subunit-interactions-predicting method, ComplexCorr, is available at http://webclu.bio.wzw.tum.de/complexcorr. © 2010 Elsevier Ltd. All rights reserved. Introduction encoded subunits that underwent accelerated amino acid replacement during the course of Subunit interactions within large protein com- primate evolution and are thus likely candidates to plexes, such as the nuclear pore and the proteasome, interact with the fast-evolving mtDNA-encoded are readily apparent from their crystal structures. subunits.13 Since cytonuclear subunit interactions However, the three-dimensional structures of many play important roles in disease and evolution,14,15 biologically important protein complexes have yet we sought to decipher such direct interactions to be resolved, thus limiting our understanding of within complex I. Here, we applied combined their subunit interactions and functionality. One evolutionary and experimental approaches to ana- such protein complex is the membrane-bound lyze the interaction of the fast-evolving nDNA- oxidative phosphorylation (OXPHOS) complex encoded subunits NDUFC2 and NDUFA1 with the NADH–ubiquinone oxidoreductase (complex I).1 mtDNA-encoded subunits of complex I. Complex I, the first and largest of the OXPHOS While coevolving amino acid residues were initially complexes (45 subunits in mammals), is the most used to predict intramolecular contacts,16 several common mutational target for mitochondrial groups have recently attempted to employ this dysfunction.2 This protein complex increased almost approach to predict residue contacts at the interface threefold in size from the so-called 14 ‘core’ subunits of interacting proteins. It has been suggested that the in Escherichia coli to 45 subunits in Homo sapiens by spatial distances between coevolving residue pairs gradual recruitment of subunits throughout harbored by interacting proteins are significantly evolution.3 Unlike other OXPHOS complexes, clues smaller than the distances between random residue for subunit interactions within the L-shaped mam- pairs.17,18 Analysis of correlated mutations assisted in malian complex I exist for the mitochondrial matrix the successful identification of interdomain or inter- arm, but not for the membrane arm of the complex.4 protein docking configurations.19,20 Moreover, coe- Hence, much of the knowledge on subunit interac- volving amino acids were found to be prevalent tions and composition within the membrane arm of among interacting residue pairs within and between the complex originates from the investigation of its proteins (‘in silico two-hybrid system’).21 Taken bacterial ortholog, NDH1.5,6 Furthermore, studies of together, these findings indicate that although the complex I assembly have revealed subunit compo- coevolution signal may not be sufficient to discern sition within subcomplexes. Still, such findings only actual contacting residue pairs, it may be informative offer general clues for specific interactions (Vogel for identifying physically interacting protein pairs. et al.7 and references within). Since understanding Hence, residue coevolution constitutes a promising subunit interactions within complex I is important approach to assessing protein–protein interactions for deciphering its function and since disruption within large complexes such as OXPHOS complex I. of complex I assembly causes diseases,7 alterna- Here, using a data set of 10 multisubunit protein tive approaches are needed. complexes with resolved crystal structures, we have Four of the five OXPHOS complexes (i.e., com- detected a clear correlation between the presence of plexes I, III, IV, and V) are composed of subunits highly coevolving residues and physical interactions encoded by nuclear and mitochondrial genomes between subunits. This enabled us to extract optimal [nuclear DNA (nDNA) and mitochondrial DNA parameters and to define the criteria for predicting (mtDNA), respectively] that differ 10 times in terms candidate interactions among mtDNA-encoded and of mutational rates.8 Accordingly, elevated amino nDNA-encoded subunits of complex I. We applied acid replacement rates indicating positive selection these criteria to all of the mtDNA-encoded subunits have been identified in nDNA-encoded subunits of complex I and two candidate nDNA-encoded that closely interact with fast-evolving mtDNA- subunits (NDUFC2 and NDUFA1) thought to encoded subunits within OXPHOS complexes with interact with them.13 This approach predicted experimentally determined three-dimensional struc- interactions between NDUFC2 and two OXPHOS ture, namely complex III, complex IV, and part of complex I mtDNA-encoded subunits (ND4 and – complex V.9 12 In a recent rigorous sequence ND5), interactions between NDUFA1 and three analysis, we identified three complex I nDNA- mtDNA-encoded subunits (ND1, ND4, and ND5), 160 Coevolution in Oxidative Phosphorylation Complex I Table 1. Complexes with known crystal structures used for an analysis of coevolving residues Protein Data Possible Bank ID Description Chains pairsa Alignmentsb Interactionsc 1GW5 AP2 clathrin adaptor core A|B|M|S 6 6 6 1W63 AP1 clathrin adaptor core A|B|M|S 6 6 6 2BCJ Gq-GRK2-G complex A|B|G|Q 6 6 3 2J0S Exon junction complex A|C|D|T 6 3 3 1LDK Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase A|B|C|D|E 10 10 5 complex 2CV5 Nucleosome core particle A|B|G|H 6 6 5 2CK3 F1-ATPase A|D|G|H|I 10 6 4 1SFC Synaptic fusion complex A|B|C|D 6 6 6 1BGY Cytochrome bc1 complex A|B|C|D|E|F|G|H|I|J 45 34 18 1V54 Cytochrome c oxidase A|B|C|D|E|F|G|H|I|J| 66 15 10 K|L a Number of possible protein pairs consisting of two different subunits for the given complex. b Subset of protein pairs in a concatenated alignment with at least 10 sequences. Only these protein pairs could be used for correlated mutation analysis. c Number of protein pairs in a concatenated alignment with at least 10 sequences and at least one residue contact.