Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments Jiapeng Zhua, Martin S. Kinga, Minmin Yub, Liron Klipcana, Andrew G. W. Leslieb, and Judy Hirsta,1 aMedical Research Council Mitochondrial Biology Unit, Cambridge, CB2 0XY, United Kingdom; and bMedical Research Council Laboratory of Molecular Biology, Cambridge, CB2 0QH, United Kingdom Edited by Hartmut Michel, Max Planck Institute of Biophysics, Frankfurt, Germany, and approved August 14, 2015 (received for review May 29, 2015) Mitochondrial complex I (proton-pumping NADH:ubiquinone oxido- involved in regulation, homeostasis, mitochondrial biogenesis, or reductase) is an essential respiratory enzyme. Mammalian complex I protection against oxidative damage (2, 5, 7, 8). contains 45 subunits: 14 conserved “core” subunits and 31 “super- Work to determine the structure of bacterial complex I cul- numerary” subunits. The structure of Bos taurus complex I, deter- minated in the 3.3-Å resolution structure of intact Thermus mined to 5-Å resolution by electron cryomicroscopy, described the thermophilus complex I (9–11). The bacterial structures revealed structure of the mammalian core enzyme and allowed the assign- many catalytically important elements, including the flavin ment of 14 supernumerary subunits. Here, we describe the 6.8-Å mononucleotide that oxidizes NADH, the iron–sulfur (FeS) resolution X-ray crystallography structure of subcomplex Iβ,alarge clusters that transfer electrons from the flavin to ubiquinone, the portion of the membrane domain of B. taurus complex I that con- ubiquinone-binding site, and elements of the proton transfer tains two core subunits and a cohort of supernumerary subunits. By apparatus: four antiporter-like structures in the membrane, and comparing the structures and composition of subcomplex Iβ and a long transverse helix that appears to strap the membrane do- complex I, supported by comparisons with Yarrowia lipolytica com- main together. These features are closely conserved in the me- plex I, we propose assignments for eight further supernumerary dium resolution structures of the complexes from B. taurus (2) subunits in the structure. Our new assignments include two (4UQ8.pdb) and the yeast Yarrowia lipolytica (12) (4WZ7.pdb). CHCH-domain containing subunits that contain disulfide bridges be- Furthermore, in the 5-Å resolution electron cryomicroscopy tween CX9C motifs; they are processed by the Mia40 oxidative-fold- (cryo-EM) structure of the B. taurus enzyme, 18 supernumerary ing pathway in the intermembrane space and probably stabilize the transmembrane helices (TMHs) were observed, and it was pos- membrane domain. We also assign subunit B22, an LYR protein, to sible to propose assignments and partial models for 14 super- the matrix face of the membrane domain. We reveal that subunit numerary subunits (2). One of these assignments (for the 13-kDa B22 anchors an acyl carrier protein (ACP) to the complex, replicating subunit) was confirmed recently in Y. lipolytica complex I using the LYR protein–ACP structural module that was identified previ- the anomalous diffraction from a bound zinc ion (13). Currently, ously in the hydrophilic domain. Thus, we significantly extend 17 supernumerary subunits remain unassigned, and 11 super- knowledge of how the mammalian supernumerary subunits are numerary TMHs in the cryo-EM model have no subunits arranged around the core enzyme, and provide insights into their assigned to them. roles in biogenesis and regulation. Fractionation of B. taurus complex I into subcomplexes Iα,Iλ, and Iβ has provided a wealth of information on its protein CHCH domain | electron transport chain | LYR protein | mitochondria | composition, and enabled subunits to be assigned to its major NADH:ubiquinone oxidoreductase Significance n mammalian mitochondria, respiratory complex I (NADH: BIOCHEMISTRY Iubiquinone oxidoreductase) (1, 2) contains 45 subunits with a Mitochondrial complex I (proton-pumping NADH:ubiquinone – combined mass of 1 MDa (3 6). Fourteen core subunits, con- oxidoreductase) is the largest respiratory chain enzyme. Mam- served from bacteria to humans, constitute the minimal complex I malian complex I contains 45 subunits: the structures of the 14 and catalyze the energy transducing reaction: NADH oxidation “core” subunits (which are sufficient for catalysis and conserved and ubiquinone reduction coupled to proton translocation across from bacteria to humans) were described in the 5-Å resolution the inner membrane. Complex I is thus critical for mammalian structure of Bos taurus complex I, but only 14 supernumerary metabolism: it oxidizes the NADH generated by the tricarboxylic subunits could be located. Here, we exploit new structural in- β acid cycle and -oxidation, produces ubiquinol for the rest of the formation from the membrane domain of mammalian complex I respiratory chain, and contributes to the proton-motive force that to assign eight further supernumerary subunits. We locate two supports ATP synthesis and transport processes. Seven of the core oxidatively-folded CHCH-domain subunits in the intermembrane subunits are hydrophilic proteins that are encoded in the nucleus space, and reveal a second LYR protein–acyl carrier protein and imported to mitochondria, and the other seven are hydropho- module. Thus, we extend knowledge of how the supernumerary bic, membrane-bound proteins (known as the ND subunits) that are subunits are arranged around the core, and provide insights into encoded by the mitochondrial genome. These two sets of seven their roles in biogenesis and regulation. subunits form the two major domains of complex I, in the matrix and inner membrane, which confer its characteristic L shape. Author contributions: J.Z., M.S.K., and J.H. designed research; J.Z., M.S.K., and M.Y. per- The protein composition of complex I from Bos taurus heart formed research; J.Z., L.K., A.G.W.L., and J.H. analyzed data; and J.H. wrote the paper. mitochondria has been characterized extensively, and is the The authors declare no conflict of interest. blueprint for the human enzyme (3–6). In addition to the 14 core This article is a PNAS Direct Submission. subunits it contains 31 supernumerary subunits (including two Data deposition: The coordinates and structure factors for subcomplex Iβ have been de- copies of the acyl-carrier protein, SDAP; ref. 2). Several super- posited with the Protein Data Bank (accession no. 5COD). numerary subunits are homologous to proteins of known func- 1To whom correspondence should be addressed. Email: [email protected]. tion and some are required for the assembly/stability of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex, but the roles of most of them are unknown: they may be 1073/pnas.1510577112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1510577112 PNAS | September 29, 2015 | vol. 112 | no. 39 | 12087–12092 Downloaded by guest on October 2, 2021 domains (3, 5, 14). Subcomplex Iλ contains all of the redox co- (Fig. 2; note that we refer to unassigned TMHs and helices by factors and represents the hydrophilic domain; subcomplex Iα their labels in the cryo-EM structure of complex I, 4UQ8.pdb; contains subcomplex Iλ plus additional subunits from the inter- ref. 2). On the left side, two TMHs were observed on the outside domain region; and subcomplex Iβ constitutes the distal section of the transverse helix in the intact enzyme (TMHs l and m) (2) of the membrane domain. Subcomplex Iβ represents the domain of but only half of TMH l (below the transverse helix) is observed in complex I that is the least-well-characterized structurally because it the subcomplex, indicating that TMH m and the upper part of contains many unassigned supernumerary subunits (2) (Table 1). TMH l become detached from the core when the transverse helix Here, we present the 6.8-Å resolution structure of subcomplex Iβ dissociates. Two additional TMH-like densities (in red in Fig. 2) from B. taurus complex I, determined by X-ray crystallography. By are also observed in the subcomplex, and form crystal contacts comparing the structures of subcomplex Iβ and intact complex I, between molecules in the asymmetric unit (Fig. S2). They were supported by comparisons of structural elements present in the not observed in the density map of complex I (2) so may be B. taurus and Y. lipolytica complexes, we propose assignments for disordered in the intact enzyme used for cryo-EM. Alternatively, a further eight mammalian supernumerary subunits. they may be artifacts from reorganization of the structure around the dissociated transverse helix, or from the cocrystallization of Results dissociated subunits from outside of subcomplex Iβ. Two addi- The Structure of Membrane–Domain Subcomplex Iβ of Complex I. Fig. tional helices on the matrix face, and four short helices on the B. taurus β intermembrane space (IMS) face, have also been added to the 1 presents the structure of subcomplex I and the re- β lationship between subcomplex Iβ and complex I. The compo- model for subcomplex I ; they were not included in the cryo-EM sition of the crystallized subcomplex was confirmed by mass model of complex I but weak density corresponding to them is visible in the density map (2). All of the new structural elements spectrometry analyses on a sample of washed and solubilized added to the model for subcomplex Iβ are shown in red in Fig. 1B. crystals; all of the 15 expected subunits of subcomplex Iβ (5, 14) except core subunit ND4 were detected. Two core subunits, ND4 Assignments for Four TMH-Containing Subunits. The conservation of B (orange in Fig. 1 ) and the first 15 TMHs of ND5 (magenta in subunits (Table 1) (5, 8) and TMHs (Fig. 2) between complex I B Fig. 1 ), were readily identified in the electron density map and subcomplex Iβ, supported by comparisons between the (shown in Fig. S1) of the subcomplex. The C-terminal region of B. taurus (2) and Y.