Sequence and Structure Comparison of ATP Synthase F0 Subunits 6 and 8 in Notothenioid Fish
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bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426195; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Sequence and structure comparison of ATP synthase F0 2 subunits 6 and 8 in notothenioid fish. 3 4 Gunjan Katyal1, Brad Ebanks1, Magnus Lucassen2, Chiara Papetti3 and Lisa 5 Chakrabarti1,4*. 6 7 8 9 10 11 12 1 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, 13 LE12 5RD, UK. 14 2 Alfred Wegener Institute, Bremerhaven, Germany. 15 3 Biology Department, University of Padova, via U. Bassi, 58/b, 35121, Padova, Italy. 16 4 MRC-Versus Arthritis Centre for Musculoskeletal Ageing Research, UK. 17 * Corresponding author [email protected] 18 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426195; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 19 20 21 22 23 24 25 Abstract: 26 27 The Channichthyidae family (icefish) are the only known vertebrate species to be 28 devoid of haemoglobin. Mitochondrial changes such as tight coupling of the 29 mitochondria have facilitated sustained oxygen and respiratory activity in the fish. This 30 makes it important to appreciate features in the sequence and structure of the proteins 31 directly involved in proton transport, which could have physiological implications. ATP 32 synthase subunit a (ATP6) and subunit 8 (ATP8) are proteins that function as part of 33 the F0 component (proton pump) of the F0F1complex. Both are encoded by the 34 mitochondrial genome and involved in oxidative phosphorylation. To explore 35 mitochondrial sequence variation for ATP6 and ATP8 we have gathered sequences 36 and predicted structures of these two proteins of fish from the Notothenioidei sub- 37 order, a sub-Antarctic species. We compared these with seven other vertebrate 38 species in order to reveal whether there might be physiologically important differences 39 that can help us to understand the unique biology of the icefish. 40 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426195; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 41 Introduction 42 43 The oceans which surround Antarctica, and their sub-zero temperatures, are home to 44 fish of the suborder Notothenioidei - a prime example of a marine species flock. This 45 suborder comprises four families, three of which include only sub-Antarctic species 46 (Bovichtidae, Pseudaphritidae, Eleginopsidae) and the Nototheniidae that 47 encompasses all Antarctic and secondary non-Antarctic species, and are sometimes 48 referred as Cryonotothenioidea[1–3]. 49 Notothenioids are renowned for their physiological adaptations to cold temperatures. 50 This includes the ability to synthesise antifreeze glycoproteins (AFGP) and antifreeze- 51 potentiating proteins (AFPP)[4]. The capacity to synthesise antifreeze glycopeptides 52 (AFGPs) is a biochemical adaptation that enabled the Notothenioidei to colonize and 53 thrive in the extreme polar environment[5]. These proteins are largely composed of a 54 Thr-Ala-Ala repeat with a conjugated disaccharide via the hydroxyl group of the Thr 55 residue and reduce the freezing point of the animals internal fluids[6,7]. 56 Within the Nototheniidae family the subfamily Channichthyinae, also known as icefish, 57 are remarkable due to the absence of haemoglobin and, in some species, myoglobin 58 too[8–10]. The sub-zero temperatures of the water they inhabit allow the highest levels 59 of oxygen solubility, which is suggested to facilitate their survival despite the loss of 60 globin proteins[10]. 61 Myoglobin is absent in the oxidative skeletal muscle in all icefish, but the absence of 62 myoglobin in cardiac muscle has been reported in only six of the sixteen species of 63 the Channichthyinae subfamily[11,12]. While the molecular genetics of how myoglobin 64 expression has been lost have been studied, the physiological differences between 65 those that express and those that do not express myoglobin are not fully understood. 66 Small intracellular diffusion distances to mitochondria and a greater percentage of cell 67 volume occupied by mitochondria are two evolutionary adaptations that can 68 compensate for the absence of myoglobin[13,14]. In the particular case of 69 Champsocephalus gunnari, the mRNA transcript of myoglobin is present in the cardiac 70 tissue but a 5-bp frameshift insertion hinders the synthesis of protein from the mRNA 71 transcript[11,15]. 72 Notothenioidei have high densities of mitochondria in muscle cells, versatility in 73 mitochondrial biogenesis and a unique lipidomic profile[16–18]. These features have 74 also been hypothesised to facilitate sustained oxygen consumption and respiratory 75 activity in the absence of haemoglobin and myoglobin. 76 Complex V of the electron transport chain, ATP synthase, is responsible for the 77 production of intracellular ATP from ADP and inorganic phosphate. Composed of an 78 F0 and F1 component, the F0 component is responsible for channelling protons from 79 the intermembrane space across the inner mitochondrial membrane and into the 80 mitochondrial matrix[19–21]. The rotation of the c-ring in F0, and with this the γ-subunit 81 of the central stalk, facilitates the translocation of protons across the inner 82 mitochondrial membrane that ultimately drives the catalytic mechanism of the F1 83 component[22,23]. 84 The motor unit F0, embedded in the inner membrane of mitochondria, is composed of 85 subunits b, OSCP (oligomycin sensitivity conferring protein), d, e, f, g, h, i/j, k which 86 are encoded by nuclear genes and subunits a (ATP6) and 8 (ATP8), which are 87 encoded by mitochondrial genes[24]. Despite the structure of the complex having 88 been first resolved decades ago, and hypotheses of the chemical mechanism were 89 developed over half a century ago, significant breakthroughs continue to be made in bioRxiv preprint doi: https://doi.org/10.1101/2021.01.11.426195; this version posted January 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 90 our understanding of both the structure and function of the enzyme and its F0 91 component[25–28]. 92 Both ATP synthase subunit a (ATP6) and subunit 8 (ATP8) are proteins that function 93 as part of the F0 component of ATP synthase, encoded by genes that overlap within 94 the mitochondrial genome[29]. This overlap is over a short, but variable between 95 species, base pair sequence where the translation initiation site of subunit 8 is 96 contained within the coding region of subunit 6. 97 The peripheral stalk is a crucial component of the F0 component forming a physical 98 connection between the membrane sector of the complex and the catalytic core. It 99 provides flexibility, aids in the assembly and stability of the complex, and forms the 100 dimerization interface between ATP synthase pairs[30]. ATP8 is an integral 101 transmembrane component of the peripheral stalk, serving an important role in the 102 assembly of the complex[31]. The C-terminus of ATP8 extends 70 Å from the surface 103 of the makes contacts with subunits b, d and F6, while the N-terminus has been 104 reported to make connections with subunits b, f and 6 in the intermembrane 105 space[32,33]. Subunit 8 is also known to play a role in the activity of the enzyme 106 complex[34]. 107 ATP6 is an α-helical protein embedded within the inner mitochondrial membrane and 108 it interacts closely with the c-ring of F0, providing aqueous half-channels that shuttle 109 protons to and from the rotating c-ring[20,35]. It has previously been reported that 110 ATP6 has at least five hydrophobic transmembrane spanning α helices domain, where 111 two of the helices h4 and h5 are well conserved across manyspecies[36]. 112 Proteins coded by mitochondrial DNA (mtDNA) are involved in oxidative 113 phosphorylation and can directly influence the metabolic performance of this pathway. 114 Evaluating the selective pressures acting on these proteins can provide insights in 115 their evolution, where mutations in the mtDNA can be favourable, neutral or harmful. 116 The amino acid changes can cause inefficiencies in the electron transfer chain, 117 causing oxidative damage by excess production of reactive oxygen species and 118 eventually interrupting the production of mitochondrial energy. Due to the tight 119 coupling of icefish mitochondria relative to their red-blooded relatives, any changes in 120 the structure of ATP Synthase subunits, particularly those directly involved in the 121 transport of protons across the membrane, could result in significant physiological 122 outcomes[37]. 123 In this work, we combine sequence analyses and secondary structure prediction 124 analyses to explore mitochondrial genetic variation for ATP6 and ATP8 in the 125 Notothenioidei suborder species Champsocephalus gunnari (C. gunnari), 126 Chionodraco rastrospinosus (C. rastrospinosus), Chaenocephalus aceratus (C. 127 aceratus), Notothenia coriiceps (N. coriiceps), Trematomus bernacchii (T. bernacchii), 128 the sub-Antarctic Eleginops maclovinus (E. maclovinus), and compare this with seven 129 other vertebrate species.