Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

Abstract

Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membraneembedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.cryo-EM | evolutionary covariance | V-ATPase | V/A-ATPase | structure V acuolar H + -ATPases (V-ATPases) are large membrane protein complexes responsible for the acidification of intracellular compartments in eukaryotes. These complexes are essential for processes including receptor-mediated endocytosis, coupled transport, and lysosomal degradation (1). V-ATPases localize to the plasma membrane of some specialized cells where they participate in bone resorption by osteoclasts (2) and urine acidification by the α-intercalated cells of the kidney (3). Malfunction or misregulation of these enzymes results in numerous disorders, including osteopetrosis (4) and renal tubular acidosis (5, 6). Expression of V-ATPases on the surfaces of some tumor cells results in increased tumor invasion and metastasis (7). The V/A-ATPase from the thermophilic eubacterium Thermus thermophilus is homologous to the eukaryotic V-ATPase and has a similar overall structure, but is smaller and simpler and functions in vivo as an ATP synthase. V-and V/A-ATPases have similar subunit folds and arrangements of subunits (8-11). The simplified architecture and superior stability of the T. thermophilus V/A-ATPase make it an ideal model to study the structure and function of V-ATPases. Both enzymes are composed of a soluble V 1 catalytic region and a membrane-embedded V O region. The V/A-ATPase subunits I, L, and C in T. thermophilus are homologous to the a, c, and d subunits in eukaryotic V-ATPases, respectively, and, for clarity, the eukaryotic subunit names are used here. With this convention, the T. thermophilus V/A-ATPase contains subunits A 3 B 3 DE 2 FG 2 ac 12 d whereas the eukaryot...

Molecular mechanisms of cutis laxa– and distal renal tubular acidosis–causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4

“…Structural modeling of human a4 subunitHomology modeling of the integral membrane domain of the human a4 subunit was generated by SWISS-MODEL, using the yeast a subunit ortholog, Vph1p, as a template (PDB:5I1M) (23,43). Subsequently, the model was corrected and the 3D representation was generated using the 3D graphical YASARA interface (44).…”

“…In an attempt to address this, we constructed a homology model for the CTa domain of the human a4 subunit, based on a recent model for the CTa domain of yeast Vph1p. The latter was built based on low-resolution X-ray crystallography, high-resolution cryo-EM, mutagenesis studies, and analysis of evolutionary covariance (23). This model showed the locations of highly conserved, key functional residues within the proton translocation pathway, or proton channel.…”

“…A, homology model for C-terminal integral membrane (CTa) domain of the human a4 subunit. This model was constructed based on the recent high-resolution cryo-EM structure and evolutionary covariance analysis for the yeast a subunit, Vph1p (23). The indicated residues are highly conserved and essential for proton translocation.…”

“…Despite such important implications for a subunit functions in disease, the structures of human a subunit isoforms are still controversial because of a lack of high-resolution structural data. Recently, however, a 6.4-Å model of the membraneintegrated domain of the yeast a subunit (Vph1p) has been published (23,24). This model is based on a synthesis of data derived from cryo-EM 3D reconstruction, evolutionary covariance mapping of key residues, and low resolution X-ray crystallography.…”

Recent developments in theCCP-EMsoftware suite

“…This library facilitates the inclusion of additional structure restraints inferred from deep-sequencing data, based on contact predictions made by external programs and provided in one of a number of data formats. Initial efforts using this approach for guiding models into cryo-EM density have proved successful (Schep et al, 2016).…”