Structure-based Analysis of High Pressure Adaptation of α-Actin

Deep-sea fishes occur to depths of several thousand meters, and at these abyssal depths encounter pressures that shallower living fishes cannot tolerate. Tolerance of abyssal pressures by deep-sea fish is likely to depend in part on adaptive modifications of proteins. However, the types of structural modifications to proteins that allow function at high pressure have not been discovered. To elucidate the mechanisms of protein adaptation to high pressure, we cloned the α-skeletal actin cDNAs from two abyssal Coryphaenoides species, C. armatus and C. yaquinae, and identified three amino acid substitutions, V54A or L67P, Q137K, and A155S, that distinguish these abyssal actins from orthologs of α-actin from non-abyssal Coryphaenoides. These substitutions, Q137K and A155S, prevent the dissociation reactions of ATP and Ca2+ from being influenced by high pressure. In particular, the lysine residue at position 137 results in a much smaller apparent volume change in the Ca2+ dissociation reaction. The V54A or L67P substitution reduces the volume change associated with actin polymerization and has a role in maintaining the DNase I activity of actin at high pressure. Together, these results indicate that a few amino acid substitutions in key functional positions can adaptively alter the pressure sensitivity of a protein. Deep-sea fishes occur to depths of several thousand meters, and at these abyssal depths encounter pressures that shallower living fishes cannot tolerate. Tolerance of abyssal pressures by deep-sea fish is likely to depend in part on adaptive modifications of proteins. However, the types of structural modifications to proteins that allow function at high pressure have not been discovered. To elucidate the mechanisms of protein adaptation to high pressure, we cloned the α-skeletal actin cDNAs from two abyssal Coryphaenoides species, C. armatus and C. yaquinae, and identified three amino acid substitutions, V54A or L67P, Q137K, and A155S, that distinguish these abyssal actins from orthologs of α-actin from non-abyssal Coryphaenoides. These substitutions, Q137K and A155S, prevent the dissociation reactions of ATP and Ca2+ from being influenced by high pressure. In particular, the lysine residue at position 137 results in a much smaller apparent volume change in the Ca2+ dissociation reaction. The V54A or L67P substitution reduces the volume change associated with actin polymerization and has a role in maintaining the DNase I activity of actin at high pressure. Together, these results indicate that a few amino acid substitutions in key functional positions can adaptively alter the pressure sensitivity of a protein. The deep-sea is typified by low temperature (1–4 °C), extremely high hydrostatic pressure, a lack of sunlight and a relatively low influx of utilizable organic material derived from primary production in surface waters. Among such environmental factors, hydrostatic pressure is thought to have the most influence on the vertical distribution of organisms and speciation in deep-sea (1France S.C. Mar. Biol. 1994; 118: 67-77Crossref Scopus (42) Google Scholar, 2France S.C. Kocher T.D. Mar. Biol. 1996; 126: 633-643Crossref Scopus (144) Google Scholar, 3Morita T. Mol. Phylogenet. Evol. 1999; 13: 447-454Crossref PubMed Scopus (46) Google Scholar). Hydrostatic pressure increases by approximately 0.1 megapascal (MPa) with every 10 m of depth in the ocean (4Saunders P.M. Fofonoff N.P. Deep-Sea. Res. 1976; 23: 109-111Google Scholar) and influences organism functions, especially those involving the formation of protein complexes, e.g. enzyme-substrate or protein-protein interaction (5Somero G.N. Am. Zool. 1990; 30: 123-135Crossref Scopus (19) Google Scholar, 6Gibbs A. Randall D.J. Farrell A.P. Deep-sea Fishes. Academic Press, New York1997: 239-278Google Scholar). Many previous studies have identified proteins from deep-sea fish that function at high pressure (5Somero G.N. Am. Zool. 1990; 30: 123-135Crossref Scopus (19) Google Scholar, 6Gibbs A. Randall D.J. Farrell A.P. Deep-sea Fishes. Academic Press, New York1997: 239-278Google Scholar, 7Somero G.N. Annu. Rev. Physiol. 1992; 54: 557-577Crossref PubMed Scopus (278) Google Scholar, 8Siebenaller J.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 1. Elsevier Press, Amsterdam1991: 323-343Google Scholar, 9Siebenaller J.F. Murray T.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 5. Elsevier Press, Amsterdam1995: 147-174Google Scholar, 10Yancey P.H. Fyfe-Johnson A.L. Kelly R.H. Walker V.P. Aunon M.T. J. Exp. Zool. 2001; 289: 172-176Crossref PubMed Scopus (92) Google Scholar), and hypothetical models for protein adaptation to deep-sea pressure have been proposed (5Somero G.N. Am. Zool. 1990; 30: 123-135Crossref Scopus (19) Google Scholar); however, the primary structures of these unique proteins have not yet been determined. Marine fish belonging to the genus Coryphaenoides known as rattails or grenadiers have been studied extensively as an excellent model in which to elucidate adaptation to the deep sea (5Somero G.N. Am. Zool. 1990; 30: 123-135Crossref Scopus (19) Google Scholar, 6Gibbs A. Randall D.J. Farrell A.P. Deep-sea Fishes. Academic Press, New York1997: 239-278Google Scholar, 7Somero G.N. Annu. Rev. Physiol. 1992; 54: 557-577Crossref PubMed Scopus (278) Google Scholar, 8Siebenaller J.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 1. Elsevier Press, Amsterdam1991: 323-343Google Scholar, 9Siebenaller J.F. Murray T.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 5. Elsevier Press, Amsterdam1995: 147-174Google Scholar, 10Yancey P.H. Fyfe-Johnson A.L. Kelly R.H. Walker V.P. Aunon M.T. J. Exp. Zool. 2001; 289: 172-176Crossref PubMed Scopus (92) Google Scholar, 11Smith Jr., K.L. Nature. 1978; 274: 362-364Crossref Scopus (95) Google Scholar, 12Marshall N.B. Developments in the Deep-Sea Biology. Blandford Press, London1979Google Scholar), because of their widespread bathymetric distribution up to a depth of about 6000 m (13Iwamoto T. Stein D.L. Occas. Pap. Calif. Acad. Sci. 1974; 111: 1-79Google Scholar). An important parameter to study in the context of pressure is the change in volume that accompanies such events as protein-ligand interactions and protein-protein interactions, for the sign and magnitude of the reaction volume change determine the reaction's sensitivity to pressure. Swezey and Somero (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar) have investigated the volume change (δV) that is associated with the polymerization of G-actin to F-actin α-skeletal actin from C. armatus (abyssal species) and C. acrolepis (non-abyssal species). The δV of actin from C. armatus was much smaller, which is advantageous for a deep-sea habitat, than that from C. acrolepis (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar). Actin is the main component of the microfilament system in all eukaryotic cells and plays a central role in maintaining the cytoskeletal structure, cell motility, cell division, intracellular movements, and contractile processes (15Sheterline P. Clayton J. Sparrow J. Actin. 4th ed. Oxford University Press, New York1999Google Scholar, 16Pollard T.D. Blanchoin L. Mullins R.D. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 545-576Crossref PubMed Scopus (1182) Google Scholar). It is one of the most conserved proteins in eukaryotic cells, for example, α-skeletal actin proteins in carp and rat share 99.4% homology at the amino acid sequence level (17Watabe S. Hirayama Y. Imai J. Kikuchi K. Yamashita M. Fish. Sci. (Tokyo). 1995; 61: 998-1003Crossref Scopus (30) Google Scholar, 18Collins J.H. Elzinga M. J. Biol. Chem. 1975; 250: 5915-5920Abstract Full Text PDF PubMed Google Scholar). It is therefore surprising that differences in the δV of this highly conserved protein have been found between two species of Coryphaenoides that inhabit different niches. In this study, we have cloned and sequenced the α-skeletal actin cDNAs from two abyssal Coryphaenoides, C. armatus and C. yaquinae, of which C. yaquinae inhabits greater depths. These actins contain three unique amino acid substitutions compared with the previously sequenced α-skeletal actin from two non-abyssal Coryphaenoides, C. acrolepis and C. cinereus (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). Biochemical analyses of the α-actin molecules purified from the skeletal muscles of C. armatus, C. yaquinae, C. acrolepis, carp, and chicken show that these amino acid substitutions are responsible for the adaptation of α-actin to high pressures in abyssal species. Here we describe, for the first time, the mechanism of adaptation of deep-sea fishes to high pressures at the amino acid sequence level. Materials—C. acrolepis, C. armatus, and C. yaquinae were collected in large live-traps made from netting material by the R/V Soyo-maru of the National Research Institute of Fisheries Science. The sampling locations were 41–40.20′ N, 142–57.40′ E, 180 m for C. acrolepis (habitat depth, about 180–2000 m) and 44–00.70′ N, 145–22.20′ E, 3940 m for C. armatus (habitat depth, about 2700–5000 m), and 39–58.10′ N, 154–59.50′ E, 5600 m for C. yaquinae (habitat depth, about 4000–6400 m). Carp (Cyprinus carpio) and chicken samples were purchased from local stores. All samples were stored below –80 °C until use. Actin Protein—Actin was isolated from the skeletal muscle of each species according to Spudich and Watt (20Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) and purified by gel-filtration chromatography over Sephadex G-200 in G buffer (0.2 mm ATP, 0.2 mm CaCl2, 0.5 mm β-mercaptoethanol, Tris-HCl, pH 7.8). Actins were converted into the Mg2+-G form as described previously (21Chen X. Rubenstein P.A. J. Biol. Chem. 1995; 270: 11406-11414Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 22Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (601) Google Scholar). Mg2+-G-actin was used immediately after its conversion from Ca2+-G-actin. Actins were stored in G buffer at 4 °C after purification and used within 3 days. The concentration of G-actin was determined spectrophotometrically using an absorption coefficient of 0.63 ml/mg at 290 nm. Polymerization, Critical Concentration, and δV Assembly—Polymerization of Mg2+-actin was initiated by adding KCl and MgCl2 to final concentrations of 50 and was by with and at at 4 °C in a high pressure cell with a high pressure cell and The change in was as a function of The concentration was determined as described previously X. Rubenstein P.A. J. Cell Biol. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The volume change (δV) in of G-actin into F-actin was by the of Swezey and Somero (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar). of Actin cDNAs from C. armatus and C. armatus and C. yaquinae muscle were and α-skeletal actin cDNAs from each were cloned as described previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). of actin were using the J. 24: Google Scholar) with from the was from the of the actin The and in the J. 1986; Scholar) were used for M. Mol. Biol. Evol. Scholar). analyses with were to the of within the Molecular that α-actin from the Coryphaenoides species were into three actin actin and actin The of actin used in this study are in C. armatus C. armatus C. yaquinae C. yaquinae C. acrolepis C. acrolepis C. cinereus C. cinereus carp, C. and was used as the of Actin the of the α-actin reaction with used volume N, and were These analyses were using and actin proteins were isolated from in each species. The for were as described previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar), for the The within a of was determined to on the of the of with The and and for actin and actin were to was by with a pH of or and The of actin to actin or actin to actin were using a dissociation of Ca2+ from actin was determined by the of N, J. Biol. Chem. Full Text PDF PubMed Google Scholar). by was to and 0.2 mm and the was at and of and at 4 °C in a high pressure reaction the is the for the is the pressure, is is the and is the temperature Chem. PubMed Scopus Google Scholar). The apparent and the apparent volume change of the Ca2+ dissociation reaction were determined as described previously Chem. PubMed Scopus Google Scholar, J. J. 1975; PubMed Scopus Google Scholar). apparent of on actin was determined by ATP with a large of such that the ATP to J. Biol. Chem. Full Text PDF PubMed Google Scholar). Actin by was converted into Mg2+-G-actin and to and the was at and of and at 4 °C in a high pressure The apparent and the apparent δV in the ATP dissociation reaction were determined as described previously Chem. PubMed Scopus Google Scholar, J. J. 1975; PubMed Scopus Google Scholar). of at was using a high pressure cell at and of 290 and at 4 °C Biochemistry. PubMed Scopus Google Scholar). DNase I DNase I was as described previously L. T. 1978; Full Text PDF PubMed Scopus Google Scholar). In DNase or with G-actin at I was to a or mm Tris-HCl, pH 4 mm mm and the change in at was at °C in a high pressure DNase I activity was from the part of the of the in Actin and the polymerization of α-skeletal actin isolated from each species as a function of pressure at actin concentrations by Actins from all species carp polymerization at each carp actin at pressures of or The to in polymerization and concentrations were determined from the polymerization The for all actins with high pressure, and at pressure the polymerization was for The for chicken and non-abyssal actin species at about and than at pressure. the of actin polymerization for the two abyssal species about at The concentrations of actin with high pressure for each species The concentrations of the two abyssal actin species were than those of species at and and from 0.1 to The volume change (δV) associated with polymerization at each pressure was determined from the concentrations The two abyssal actin species a much smaller δV at each pressure than the species, in with previous (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar). The δV of actins from chicken and non-abyssal species at high pressure, those from the abyssal species These for chicken and non-abyssal species, the coefficient of for F-actin was than that for G-actin and that the by interactions was by high pressure. was such in in actins from the abyssal species. the δV of carp that of species, with high pressure, which that for carp the coefficient of for G-actin was than that for in carp G-actin is than the G-actin of the species. this carp actin was to at pressures of or and α-skeletal actin were cloned from each of the two abyssal species and compared with the two non-abyssal actin species previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). Molecular that these α-actin from the abyssal species were all as actin These were with the high the with an amino acid sequence to that of actin of the non-abyssal species was actin and the was and for C. armatus and C. yaquinae, the non-abyssal actin was actin The amino acid of actin and by one amino acid residue at position which is in actin and in actin (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). The sequence of actin from that of actin by two amino V54A or L67P and The of skeletal muscle actin that and are in a of 137 and are in the and and Nature. 1990; PubMed Scopus Google of the amino acid of actin actin actin in a of actin with ATP and and a ATP and of actin Nature. 1990; PubMed Scopus Google Scholar). The positions of substitutions found in this study are of ATP and Ca2+ in and are by The and are and These are using J. 24: Google of Actin the of the α-actin the of was investigated using was with one which different from the two about for actin and actin and about for actin to the different of their that the were the actin The results that was of the two in each species. The of actin to actin or actin to actin was for C. yaquinae, for C. armatus, and for C. acrolepis, the of protein using The of actin to actin was for C. yaquinae and for C. armatus not The protein from C. acrolepis, which have the not by this not These results of the were not by using not These of the that is for the species habitat, as described and actin from abyssal species has a lysine at position the and all actins from the species have a at this and all actin from abyssal species have a at residue Actin ATP by the ATP and between two and which to and 3 and Nature. 1990; PubMed Scopus Google Scholar, P. Science. 2001; PubMed Scopus Google Scholar). Actin a or in a deep by the and of the ATP and actin and and Nature. 1990; PubMed Scopus Google Scholar, J. Res. Cell 1992; 13: PubMed Scopus Google Scholar). actin has a for Ca2+ than for the much concentration of that the main of the in investigated the of high pressures on the and of actin protein at pressures and dissociation of actin from abyssal species were much by high pressures than those of the which at pressures greater than The dissociation of Ca2+ in abyssal actins not from that of the actins at pressure, which that the actins to Ca2+ with the the differences in the dissociation of ATP that actin ATP than as previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). The apparent δV in the Ca2+ dissociation reaction at pressures greater than was as for abyssal species and for the species. 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To of pressures on the actin structure, we the of at the not the of actins from carp, and non-abyssal species to at 10 not and in the for carp actin was than that for chicken and non-abyssal by the of the abyssal actins not change at and Actin for Ca2+ is greater than for were differences in between the and Mg2+-G-actin for all actin species not These results indicate that high pressure the of the in in the of actin which one of the that the ATP and the DNase I of the two abyssal species a V54A substitution or an L67P substitution The that these are in In I interactions, DNase I the DNase in and with and in 4 Nature. 1990; PubMed Scopus Google Scholar, J. J. Scopus Google Scholar). 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Deep-sea Fishes. Academic Press, New York1997: 239-278Google Scholar, 7Somero G.N. Annu. Rev. Physiol. 1992; 54: 557-577Crossref PubMed Scopus (278) Google Scholar, 8Siebenaller J.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 1. Elsevier Press, Amsterdam1991: 323-343Google Scholar, 9Siebenaller J.F. Murray T.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 5. Elsevier Press, Amsterdam1995: 147-174Google Scholar). results a mechanism for enzyme-substrate interactions pressure. The Q137K and substitutions indicate that actin species than abyssal actin have in the protein to the of amino acid with smaller The results from the and however, that these at high the and and previous studies show that abyssal actins form with ATP and Ca2+ than actin species (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar, R.R. Somero G.N. Biochemistry. PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar). the actin found in fishes the in the however, the of the residue at position in actin in large of in this study was collected from a of actin isolated from which we were to by the of proteins in is a the system for and in α-skeletal has been not as yet because of such as the formation of and the low of protein J. J. T. C. Acad. Sci. S. A. 1990; Scopus Google Scholar). are therefore this system for are a of proteins T.D. J.A. Annu. Rev. 1986; PubMed Google Scholar), as a actin is one of the most conserved proteins with results have that a function that species to to a can in a protein by a few amino acid substitutions in key functional the on the α-actin that actin from actin and that actin from actin by a few amino acid substitutions The previous study that fishes actin (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). the from to sea and from surface to abyssal the α-actin each These are with of protein speciation Mol. Biol. Evol. Google Scholar), the of S. by Google Scholar), and the from the on T. Mol. Phylogenet. Evol. 1999; 13: 447-454Crossref PubMed Scopus (46) Google Scholar). I most to Somero for and of the I K. of the National Research Institute of Fisheries for the to this study and abyssal I S. Y. and M. of for in the actin Research Institute of Fisheries for and the and of the R/V Soyo-maru for I an and an for on the