*Center for Molecular Medicine and Genetics
Department of Anatomy and Cell Biology, Wayne State University School of Medicine
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Abstract |
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Introduction |
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The mitochondrial genome consists of 13 genes that encode proteins utilized in oxidative production of energy. Each mitochondrial DNA (mtDNA) gene product must interact with proteins encoded in the nuclear genome to carry out its function. In total, mtDNA-encoded proteins participate in four of the five complexes that perform oxidative phosphorylation. COX is the terminal enzyme complex of the respiratory chain found in the mitochondrial inner membrane (Kadenbach et al. 1983
; Capaldi 1990
; Tsukihara et al. 1995
). Mammalian COX contains 3 subunits encoded by mtDNA and 10 encoded by nuclear DNA (nDNA). Interactions of mtDNA- and nDNA-encoded proteins at the functional level provide unique opportunities to study the evolution of protein-protein interactions and the effects of these interactions on the evolution of their respective genomes.
The presumed importance of functional interactions between mtDNA- and nDNA-encoded proteins has been experimentally confirmed. Kenyon and Moraes (1997)
determined that cybrid cells constructed with human nDNA and mtDNAs from different primate species varied in ability to survive in media that required oxidative metabolism. In those experiments, mtDNAs that were evolutionarily close to humans (chimps and gorillas) were able to functionally complement the human nDNA gene products. In contrast, cybrids constructed with more evolutionarily distant mtDNAs did not survive. Conceptually similar experiments have been performed by repeatedly backcrossing copepod Tigriopus califonicus populations, thereby placing the maternally inherited mtDNA genome of one population with the paternal nDNA of another (reviewed in Burton, Rawson, and Edmonds 1999
). COX activities of the resulting progeny were significantly lower than those of both native populations. One interpretation of these studies is that mtDNA-encoded proteins are less able to function with the nuclear counterparts of other populations or species because they disrupt protein interactions that have coevolved over time.
These studies demonstrated the functional importance of mtDNA-nDNAencoded protein interactions, but they did not explore the evolution of such interactions. In this report, a combination of evolutionary and crystallographic data from the COX holoenzyme was used to study interactions at the DNA level. Residues in close physical proximity to those of a subunit encoded by another genome are clearly functionally important. Thus, nonsynonymous mutations in codons encoding such close-contact residues are not likely to escape the scrutiny of natural selection, either in its positive form when selection for advantageous amino acid replacements spreads the replacements through a species lineage or, alternatively, in its purifying form when selection continues to favor these amino acid replacements after they have spread throughout the lineage. We found not only that the close-contact residues have different nonsynonymous substitution rates than the residues not involved in protein interactions, but also that the type of interaction differs for mtDNA- and nDNA-encoded residues.
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Materials and Methods |
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Three categories of interaction ratios are expected. An i indistinguishable from 1 would result if the evolutionary rate of the close-contact residues were the same as the evolutionary rate of the noncontact residues, indicating that the close-contact residues were under neither greater purifying selection nor greater positive selection than the noncontact residues. i < 1 would result from a reduced evolutionary rate of the close-contact residues relative to the noncontact residues, indicating that greater purifying selection but less positive selection acted on the close-contact residues than on the noncontact residues. Finally, i > 1 would be due to a more rapid nonsynonymous substitution rate for the close- contact residues, indicating that they are under less purifying selection but under more positive selection than the noncontact residues. On the one hand, we interpret i < 1 as being due to the protein's close-contact residues (those involved in protein interaction) being under greater structural-functional constraints than the protein's noncontact residues (those less likely to be involved in protein interaction). We refer to such a state as a "constraining interaction." On the other hand, we interpret i > 1 as being due to many different amino acid replacements among the close contact residues being required to optimize this protein's interaction with the other protein. We refer to such a state as an "optimizing interaction."
Since i is a comparison of nonsynonymous substitutions for independently selected residues of a protein with the evolutionary rate of the remaining residues of the same protein, the residues have a common evolutionary history. Mutation rates, population size, phylogenetic history, and other evolutionary parameters are unlikely to differ within a protein. Consequently, for the purposes of our study, relative rates of evolution rather than absolute rates are used since for any protein under study, the period of time over which the nonsynonymous substitutions occurred is the same for the close-contact residues as for the noncontact residues.
Data Sets
Crystallographic data for COX genes from bovine heart mitochondria (Tsukihara et al. 1995, 1996
) were first grouped by encoding genome. Nucleotide sequences encoding COX nDNA residues that were also in close proximity to COX mtDNA-encoded residues (fig. 1 ) were then segregated and placed in a separate data set from those not in close contact with mtDNA-encoded residues (fig. 2 ). Similarly, COX residues encoded by mtDNA were segregated on the basis of proximity to nDNA-encoded residues. Physical distances between residues were calculated with a computer program provided by Dr. Philip D. Martin. Residues
4 Å apart, the nominal upper limit for weak interactions (Martin et al. 1997
), were segregated.
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A seven-taxon data set was constructed from the mtDNA-encoded data in order to calculate i on a lineage-by-lineage basis using the codeml program of the PAML package. Tree topologies were constrained to be consistent with relationships derived from analyses of complete mitochondrial genomes (Arnason, Gullberg, and Janke 1997, 1998
; Janke, Xu, and Arnason 1997
) and morphological and other molecular data (Liu and Miyamoto 1999
). In all cases, seven-taxon data sets were used to limit computation time.
DNA Sequences
Most nucleotide sequences of COX genes were acquired from GenBank and aligned with CLUSTAL W (alignments can be obtained from the website of L.I.G., http://cmmg.biosci.wayne.edu/lgross/lgross-home.html). COX crystallography data of Tsukihara et al. (1996)
were acquired from the Brookhaven Protein Data Bank. Seven nDNA-encoded COX genes (COX4, COX5B, COX6AH, COX6B, COX7AH, COX7B, and COX7C) were analyzed with methods described below for sequences of human, cow, mouse, and rat. There were not sufficient evolutionary data to include COX5A and COX6C. COX8H was excluded from the analyses because it is absent in humans (Van Kuilenburg et al. 1988
; Rizzuto et al. 1989
). Nucleotide sequences of 26 mammalian taxa were analyzed for the mtDNA-encoded COX genes (COX1, COX2, and COX3). These mammalian taxa were Bos taurus, Balaenoptera physalus, Balaenoptera musculus, Equus caballus, Equus asinus, Rhinoceros unicornis, Ceratotherium simum, Homo sapiens, Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus, Hylobates lar, Felis catus, Halichoerus grypus, Phoca vitulina, Mus musculus, Rattus norvegicus, Erinaceus europaeus, Didelphis virginiana, Macropus robustus, Ornithorhynchus anatinus, Oryctolagus cuniculus, Myoxus glis, Cavia porcellus, and Armadillo officinalis.
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Results |
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Interaction Ratios (Phylogenetic)
An increased nonsynonymous substitution rate (or amino acid replacement rate) in primates has been demonstrated for a number of COX subunits and CYC (Baba et al. 1981
; Evans and Scarpulla 1988
; Adkins and Honeycutt 1994
; Wu et al. 1997, 2000
; Schmidt, Goodman, and Grossman 1999
). To examine whether those results were related to the observation reported here, we determined whether the optimizing interaction between the mtDNA- and nDNA-encoded subunits was emphasized in the primate lineage. Phylogenetic trees were used to apportion the nonsynonymous substitutions for the contact and noncontact mtDNA data sets, and imt_n was calculated for each lineage (fig. 3
). Most lineages showed i > 1; imt_n values of <1 were found only for short internal branches. Results of analyses using alternative tree topologies and species did not differ substantially in having an imt_n of >1, although with some topologies the primate imt_n was >4. We conclude that primates do not show a markedly different imt_n than other lineages of mammals. Instead, optimizing interactions have a broad phylogenetic distribution and are present in most mammalian lineages examined. These results suggest that (1) the co-occurring rate accelerations observed for primate COX subunits are not concentrated in the residues that have a direct interaction, and (2) the optimizing interaction is not due to the increased nonsynonymous substitution rate in primates previously observed for several subunits of COX.
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Discussion |
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The prevailing (although untested) view, which is based on the generally conservative nature of protein evolution, is that constraining interactions are typical (e.g., see Griffiths 1998
; Lockless and Ranaganthan 1999
). The calculated interaction ratios of <1 (a constraining interaction) for COX subunits encoded by nDNA are consistent with this view. In contrast, an optimizing interaction was detected for mtDNA-encoded residues of COX. These results suggest that the faster mtDNA mutation rate, which allows sampling of more residues in the interacting region, makes mtDNA the predominant partner in accommodating mutations important for subunit interaction.
It would be interesting to examine the evolution of protein interaction in other complexes of the respiratory chain that have a mixed genetic origin for a similar pattern. Moreover, the combined use of crystallographic information and evolutionary data is not limited to studies of mtDNA interactions with nDNA. For example, the methods employed in this study can also be utilized in analysis of the evolution of protein interaction within complexes from plant cells that are encoded by chloroplast DNA and nDNA. Basic requirements for an accurate calculation of i include (1) nucleotide sequences of taxa that are sufficiently divergent to allow for accurate calculation of nonsynonymous substitution rates or amino acid replacements, (2) a reasonable number of residues for each data set, and (3) crystallographic data of molecules that potentially interact. Probably the most important methodological limitation is that a large number of interacting residues are required to develop a statistically relevant sample. However, in the absence of a large sample of residues, important information can still be derived from the combined use of crystallographic and phylogenetic data for identifying residues that covary.
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Acknowledgements |
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Footnotes |
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1 Present address: Institute of Chemical Toxicology, Wayne State University
1 Keywords: coevolution
protein crystallography
phylogenetic analysis
2 Abbreviations: COX, cytochrome c oxidase; nDNA, nuclear DNA; mtDNA, mitochondrial DNA.
3 Address for correspondence and reprints: Lawrence I. Grossman, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201. l.grossman{at}wayne.edu
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literature cited |
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---|
Adkins, R. M., and R. L. Honeycutt. 1994. Evolution of the primate cytochrome c oxidase subunit II gene. J. Mol. Evol. 38:215231[ISI][Medline]
Arnason, U., A. Gullberg, and A. Janke. 1997. Phylogenetic analyses of mitochondrial DNA suggest a sister group relationship between Xenarthra (Edentata) and Ferungulates. Mol. Biol. Evol. 14:762768[Abstract]
. 1998. Molecular timing of primate divergences as estimated by two nonprimate calibration points. J. Mol. Evol. 47:718727[ISI][Medline]
Baba, M. L., L. L. Darga, M. Goodman, and J. Czeluzniak. 1981. Evolution of cytochrome c investigated by the maximum parsimony method. J. Mol. Evol. 17:197213[ISI][Medline]
Brooks, D. R. 1979. Testing the context and extent of host- parasite coevolution. Syst. Zool. 28:299307[ISI]
Burton, R. S., P. D. Rawson, and S. Edmonds. 1999. Genetic architecture of physiological phenotypes: empirical evidence for coadapted gene complexes. Am. Zool. 39:451 462[ISI]
Cann, R. L., W. M. Brown, and A. C. Wilson. 1984. Polymorphic sites and the mechanism of evolution in human mitochondrial DNA. Genetics 106:479499
Capaldi, R. A. 1990. Structure and function of cytochrome-c oxidase. Annu. Rev. Biochem. 59:569596[ISI][Medline]
Ehrlich, P. R., and P. H. Raven. 1964. Butterflies and plants: a study in coevolution. Evolution 18:586608
Evans, M. J., and R. C. Scarpulla. 1988. The human somatic cytochrome c gene: two classes of process pseudogene demarcate a period of rapid molecular evolution. Proc. Natl. Acad. Sci. USA 85:96259629
Fryxell, K. J. 1996. The coevolution of gene family trees. Trends Genet 12:364369
Griffiths, C. S. 1998. The correlation of protein structure and evolution of a protein-coding gene: phylogenetic inference using cytochrome oxidase III. Mol. Biol. Evol. 15:1337 1345
Hafner, M. S., and S. A. Nadler. 1988. Phylogenetic trees support the coevolution of parasites and their hosts. Nature 332:258259
Hughes, A. L. 1992. Coevolution of the vertebrate integrin alpha- and beta-chain genes. Mol. Biol. Evol. 9:216234[Abstract]
Janke, A., X. Xu, and U. Arnason. 1997. The complete mitochondrial genome of the wallaroo (Macropus robustus) and the phylogenetic relationship among Monotremata, Marsupialia, and Eutheria. Proc. Natl. Acad. Sci. USA 94: 12761281
Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules. Pp. 21132 in N. H. Munro, ed. Mammalian protein metabolism. Academic Press, New York
Kadenbach, B., J. Jarausch, R. Hartmann, and P. Merle. 1983. Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoresis procedure. Anal. Biochem. 129:517521[ISI][Medline]
Kenyon, L., and C. T. Moraes. 1997. Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids. Proc. Natl. Acad. Sci. USA 94:91319135
Kumar, S., K. Tamura, and M. Nei. 1993. MEGA: molecular evolutionary genetics analysis. Institute of Molecular Evolutionary Genetics, Pennsylvania State University, University Park
Li, W. H., C. I. Wu, and C. C. Luo. 1985. A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Mol. Biol. Evol. 2:150174[Abstract]
Liu, F.-G. R., and M. M. Miyamoto. 1999. Phylogenetic assessment of molecular and morphological data for eutherian mammals. Syst. Biol. 48:5464[ISI][Medline]
Lockless, S. W., and R. Ranaganthan. 1999. Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286:295299
Martin, P. D., M. G. Malkowski, J. Box, C. T. Esmon, and B. F. Edwards. 1997. New insights into the regulation of the blood clotting cascade derived from the X-ray crystal structure of bovine meizothrombin des F1 in complex with PPACK. Structure 5:16811693
Moyle, W. R., R. K. Campbell, R. V. Myers, M. P. Bernard, Y. Han, and X. Wang. 1994. Co-evolution of ligand-receptor pairs. Nature 368:251255
Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418426[Abstract]
Rizzuto, R., H. Nakase, B. Darras, U. Francke, G. M. Fabrizi, T. Mengel, F. Walsh, B. Kadenbach, S. DiMauro, and E. A. Schon. 1989. A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues. J. Biol. Chem. 264:1059510600
Schmidt, T. R., M. Goodman, and L. I. Grossman. 1999. Molecular evolution of the COX7A gene family in primates. Mol. Biol. Evol. 16:619626[Abstract]
Schmidt, T. R., S. A. Jaradat, M. Goodman, M. I. Lomax, and L. I. Grossman. 1997. Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms. Mol. Biol. Evol. 14:595601[Abstract]
Sitnikova, T., and C. Su. 1998. Coevolution of immunoglobulin heavy- and light-chain variable-region gene families. Mol. Biol. Evol. 15:617625[Abstract]
Swofford, D. L. 1998. PAUP: phylogenetic analysis using parsimony. Smithsonian Institution, Washington, D.C
Tsukihara, T., H. Aoyama, E. Yamashita, T. Tomizaki, H. Yamaguchi, K. Shinzawa-Itoh, R. Nakashima, R. Yaono, and S. Yoshikawa. 1995. Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 Å. Science 269:10691074
. 1996. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Å. Science 272:11361144
Turner, J. R. G. 1977. Butterfly mimicry: the genetical evolution of an adaptation. Evol. Biol. 10:163206
Van Kuilenburg, A. B. P., A. O. Muijsers, H. Demol, H. L. Dekker, and J. J. Van Beeumen. 1988. Human heart cytochrome c oxidase subunit VIII. Purification and determination of the complete amino acid sequence. FEBS Lett. 240:127132[ISI][Medline]
Wu, W., M. Goodman, M. I. Lomax, and L. I. Grossman. 1997. Molecular evolution of cytochrome c oxidase subunit IV: evidence for positive selection in simian primates. J. Mol. Evol. 44:477491[ISI][Medline]
Wu, W., T. R. Schmidt, M. Goodman, and L. I. Grossman. 2000. Molecular evolution of cytochrome c oxidase subunit I in primates: is there co-evolution between mitochondrial and nuclear genomes? Mol. Phylogenet. Evol. 17:294304
Yoshikawa, S., K. Shinzawa-Itoh, R. Nakashima et al. (13 co-authors). 1998. Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase. Science 280:1723 1729