Structure of subcomplex Iβ of mammalian 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 . 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 | | 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. lipolytica (12) (Fig. S3), allows us to ND5 [the transverse helix that runs along the membrane domain propose four new subunit assignments. First, we divide the plus TMH16 (2, 11)] is not observed, indicating that it dissociates TMHs into three classes. Class I contains seven TMHs from the from the core when the enzyme fragments and is disordered in cryo-EM structure of complex I that have already been assigned the crystals. This observation is consistent with the position of to subunits B9, B14.7, B16.6, and MWFE (2). None of these TMH16, anchoring the end of the transverse helix at the in- subunits have been detected in subcomplex Iβ (5), and because terface of subunits ND2 and ND4L that are not present in the they are located in a clearly different region of the complex (Fig. subcomplex. On the right side of the subcomplex, adjacent to 2, TMHs in cyan) they are not present in its structure. Class II core subunits ND4 and ND5, are six supernumerary TMHs contains four additional TMHs (c, d, e, and m) that are also conserved from the cryo-EM structure of intact complex I (2) absent completely from the structure of subcomplex Iβ, but they

Table 1. Supernumerary subunits in the membrane domain of complex I (5, 8) † B. taurus Length* H. sapiens Y. lipolytica Length* Features Subcomplex Assigned

15 kDa 105 NDUFS5 NIPM 88 2-CX9C motif 1α This work MWFE 70 NDUFA1 NIMM 86 1 TMH 1α Previously (2) B9 83 NDUFA3 NI9M 78 1 TMH 1α Previously (2)

PGIV 171 NDUFA8 NUPM 171 2 2-CX9C motifs 1α Previously (2) B14.7 141 NDUFA11 NUJM 197 4 TMHs 1α Previously (2) B16.6 144 NDUFA13 NB6M 122 1 TMH 1α and 1λ Previously (2) SDAP 88 NDUFAB1 ACPM2 92 Acyl carrier 1α and 1β Previously (2) MNLL 58 NDUFB1 (1 TMH) 1β AGGG 72 NDUFB2 1 TMH 1β B12 97 NDUFB3 NB2M 59 1 TMH 1β ‡ B15 128 NDUFB4 NB5M 93 1 TMH (1α and 1β) This work SGDH 143 NDUFB5 1 TMH 1β B17 127 NDUFB6 1 TMH 1β

B18 136 NDUFB7 NB8M 98 2-CX9C motif 1β This work ASHI 158 NDUFB8 NIAM 125 1 TMH 1β B22 178 NDUFB9 NI2M 109 LYR motif 1β This work PDSW 175 NDUFB10 NIDM 91 1β This work ESSS 125 NDUFB11 NESM 204 1 TMH 1β This work KFYI 49 NDUFC1 1 TMH This work ‡ B14.5b 120 NDUFC2 1 or 2 TMHs (1β) This work NUXM 169 2 TMHs NEBM 73 1 TMH NUNM 118 (1 TMH) NUUM 89 1 TMH

*The lengths are given for the mature proteins, for B. taurus and Y. lipolytica. † Homology relationships between the mammalian and Y. lipolytica subunits are based predominantly on the analysis of Huynen et al. (15), and are in accord with the recent summary by Kmita and Zickermann (8). ‡Subunits detected at low levels.

12088 | www.pnas.org/cgi/doi/10.1073/pnas.1510577112 Zhu et al. Downloaded by guest on October 2, 2021 A B

transverse matrix helix

toe heel IMS

Fig. 1. Comparison of the structures of subcomplex Iβ and complex I from B. taurus.(A) The density from subcomplex Iβ (red) is superimposed on that of intact B. taurus complex I (gray, semitransparent). (B) The model for subcomplex Iβ is superimposed on that for intact complex I (pale blue, 4UQ8.pdb). Core subunits ND4 and ND5 in subcomplex Iβ are in orange and magenta, respectively, supernumerary structures observed in both complex I and subcomplex Iβ are in dark blue, and new structures observed in subcomplex Iβ are in red. The figure is presented in the opposite orientation (with the “toe” pointing to the left) to subsequent figures to show the region around the transverse helix.

are located adjacent to it in intact complex I and thus in the role, or [as there are 18 TMHs observed in Y. lipolytica complex I fracture zone that cleaves apart to separate subcomplex Iα from (12) and only 16 predicted] the homolog of B14.5b may not have subcomplex Iβ (Fig. 2, complete TMHs in green). Finally, class III been identified yet. The latter suggestion is supported by the contains seven TMHs that are present in the structures of both existence of a known B14.5b homolog in Neurospora crassa (16). intact complex I and subcomplex Iβ (TMHsf,g,h,i,j,k,and In this case, NUXM can be assigned to the two TMHs that are l, shown in blue in Fig. 2), plus the two additional, puta- specific to Y. lipolytica (Fig. S3), consistent with the composition tive TMHs (Fig. 2, red) that are observed in the subcomplex. of Y. lipolytica subcomplex Iδ (17), and with the position of these Assignments of TMHs in classes II and III are described in the two TMHs adjacent to the three N-terminal TMHs of ND2 that following sections. are also absent from B. taurus (18). Specific assignments for the fracture-zone TMHs c, d, e, and In subcomplex Iβ seven subunits with single predicted TMHs m were formulated as follows. First, three TMH-containing have been detected in apparently stoichiometric levels (AGGG, subunits have been detected either in both subcomplex Iα and Iβ ASHI, ESSS, MNLL, SGDH, B12, and B17; Table 1; ref. 5). (B15, one predicted TMH), in neither (KFYI, one predicted They must account for the TMHs observed in the structure of TMH), or in subcomplex Iβ at low levels only (B14.5b, one or subcomplex Iβ, assuming that there are no as-yet-undetected two predicted TMHs) (5), suggesting they are from the fracture proteins present. If the two new TMHs (β1 and β2) are real, then zone, so we assign them to TMHs c, d, e, and m. Second, TMHs there are fewer proteins than TMHs so one or more subunits c, d, and e form a group of three TMHs that must contain the may be present in more than one copy, or have a more complex two-TMH subunit B14.5b. Therefore, the isolated TMH m is topology than predicted. Currently, there is little information to from KFYI or B15; we assign it to B15 because both TMH m and distinguish the TMHs from one another, but by comparing the subunit B15 are conserved in Y. lipolytica, whereas no KFYI structures from B. taurus (2) and Y. lipolytica (12) (Fig. S3)we homolog is known (Fig. S3; Table 1; refs. 8, 15). B15 has been propose an assignment for subunit ESSS to TMH g as follows. detected in both subcomplexes Iα and Iβ (5, 14) and TMH m is suitably positioned to remain weakly associated with the disor- BIOCHEMISTRY dered transverse helix in subcomplex Iβ, and with adjacent sub- units such as B14.7 and 42 kDa in subcomplex Iα. Indeed, (hydrophilic arm) left side B14.7 l *β1 dissociation of the transverse helix severely disrupts the local m structure in subcomplex Iβ (Fig. 1), including the loss of helix q β2 that runs across the matrix face (Fig. 3A). As subunit B15 con- * tains a 25-residue N-terminal helix we may speculate that helix q MWFE is part of B15. Third, the connectivity within the three TMHs (c, & B9 j k d, and e) in the cryo-EM density for B. taurus complex I was judged ambiguous (2), whereas TMHs d and e were modeled as a B16.6 c e g ( ) single chain in Y. lipolytica complex I (12), tentatively suggesting heel * ( )h * i toe d *f * TMH c is from KFYI and TMHs d and e are from B14.5b. This right side assignment is supported by the absence of both TMH c (12) and β Y. lipolytica Fig. 2. The transmembrane helices present in B. taurus subcomplex I and KFYI (8, 15) in , and by the peripheral location of complex I. The view is from the matrix face. Subunits ND4 and ND5 in sub- TMH c in B. taurus (2) (it interacts only with TMHs d and e, complex Iβ are in orange and magenta, respectively, except that the portion explaining why KFYI is lost upon fractionation whereas B14.5b of ND5, including the transverse helix, that is not observed in the sub- is retained in low levels; ref. 5). Finally, models for B15, KFYI, complex is in wheat. Core subunits in complex I that are not present in the and B14.5b, based on secondary structure predictions, fit well subcomplex are also shown in wheat. Supernumerary TMHs assigned pre- into the cryo-EM density (Fig. S4). For B14.5b the unequal viously that are not present in subcomplex Iβ are in cyan; additional TMHs β lengths of the two helices define TMH e as the shorter, N-ter- that are observed in the structure of complex I but not subcomplex I are in green; TMHs observed in the structures of both complex I and subcomplex Iβ minal helix. Only the identity of the protein forming TMHs d and β Y. lipolytica Y. lipolytica are in blue, and the two putative TMHs observed only in subcomplex I are in ein remains unclear because no B14.5b red. The unassigned TMHs are labeled according to their names in 4UQ8.pdb homolog is known, and only subunit NUXM (with no known with those that are not conserved in the structure of Y. lipolytica complex I B. taurus homolog) has two predicted TMHs (Table 1). NUXM marked *, and those that are present in the Y. lipolytica structure but do not and B14.5b may be different proteins fulfilling the same structural superimpose on the TMHs in the B. taurus enzyme marked (*).

Zhu et al. PNAS | September 29, 2015 | vol. 112 | no. 39 | 12089 Downloaded by guest on October 2, 2021 β A (matrix face) B22 (n) that it binds SDAP- to the membrane domain (2, 21). Here, we SDAP used the models for SDAP-α and SDAP-β to map the B14- *q SDAP-α module (from 4UQ8.pdb for B. taurus complex I) onto the matrix face of subcomplex Iβ, and found that three helices of B14 superimpose closely on three helices (chain n) in sub- β *β3 complex I , constituting an equivalent module (Fig. S5). Subunit B22, chain n, and SDAP-β are all conserved in Y. lipolytica (12) *β4 and increased density on the matrix face of ND5 in B. taurus, (hydrophilic *o relative to Y. lipolytica (Fig. S3), is probably partly due to the arm) *p longer length of the B. taurus B22 homolog. Therefore, we assign *42 kDa fracture β zone 180 the three helices (chain n) on the matrix face of subcomplex I B (IMS face) (and complex I) to subunit B22 (Figs. 3A and 4). w B16.6 * Assignments for Three Subunits on the IMS Face. Two subcomplex Iβ β5 s subunits that do not contain any TMHs have not been discussed § PGIV PDSW (t) or modeled thus far: B18 and PDSW. B18 is known to be located on the IMS face, and to contain a CHCH domain—a double CX9C domain that forms a helix-turn-helix structure with two § disulfides between the helices (22, 23). Secondary structure B18 B. taurus Y. lipolytica *β6 *β7 (u,v) predictions for both the and homologs in- *β8 dicate it is followed by a helix of more than 30 residues. There heel 15 kDa (r) toe are two candidates for B18 in the structure of subcomplex Iβ Fig. 3. The matrix and IMS faces of B. taurus complex I. (A) The hydrophilic (Fig. 3B): the two helices in chain t, and chains u and v together. arm has been removed to show the supernumerary subunits present on the Little is known about subunit PDSW: we assign it to the IMS face matrix face of complex I. (B) The supernumerary subunits present on the IMS because it is predicted to contain three helices of more than 15 face of complex I. For both panels, the structures of the supernumerary residues (including one of 35) that are difficult to accommodate subunits on the faces are in color and labeled accordingly. The structures of in helices observed on the matrix face of subcomplex Iβ but can ND4 and ND5 are in light orange with the other subunits in white and the be readily accommodated on the IMS side, and because it con- line along which the complex fractures to form subcomplex Iβ (to the right side) is indicated. The figure was created using 4UQ8.pdb for complex I (2) tains conserved Cys residues that may confer structural stability plus additional elements from the structure of subcomplex Iβ. Asterisks in- through disulfides formed in the oxidizing IMS (24). The two dicate elements that are not observed in the structure of Y. lipolytica com- helices in chain t, and chains u and v together, are thus also plex I (12) and § indicates that an alternative assignment in which the candidates for the assignment of PDSW. Although we are con- assignments for PDSW and B18 are exchanged for one another is possible. fident that B18 and PDSW together account for the four helices in chains t, u, and v, it is not currently possible to confidently Four unassigned TMHs in subcomplex Iβ (TMHs g, j, k, and l) are closely conserved in the structures of the complexes from B. taurus and Y. lipolytica (Fig. S3) and three of the unassigned TMH-containing subunits identified in subcomplex Iβ (ASHI, ESSS, and B12; Table 1) have known Y. lipolytica homologs. Therefore, ASHI, ESSS, and B12 are candidates for TMHs g, j, B8 k, and l. A domain is present on the matrix face of Y. lipolytica complex I, on the right-side adjacent to ND4 and conserved 18 kDa B. taurus TMH g (Fig. S3) that is not present in . In the model for B13 B14 Y. lipolytica 13 kDa complex I (12) TMH g is connected to one of the SDAP-α helices modeled into this domain, as well as to a conserved helix 42 kDa (helix β5 in Fig. 3B) in the IMS, which is in a position consistent SDAP-β with the antibody label to Y. lipolytica ESSS observed by EM 39 kDa (19). The assignment of ESSS to TMH g explains the extra ESSS MWFE & B9 B15 B22 density on the matrix face of Y. lipolytica complex I because the B16.6 B14.7 Y. lipolytica ESSS N-terminal domain of ∼120 residues is twice as long as in B. taurus. Furthermore, in both homologs the pre- 15 kDa dicted TMH is followed closely by a further helix of ∼20 residues B. taurus (the C-terminal domain contains 45 residues in ), which B18* is consistent with helix β5 in the IMS. In consequence, we pro- pose that TMH g is formed by subunit ESSS, with the N terminus PGIV KFYI B14.5b PDSW* on the matrix face. Fig. 4. Summary of the assignments of supernumerary subunits in B. taurus Identification of Subunit B22 on the Matrix Face. Two copies of complex I. A semitransparent surface for the density map for mammalian SDAP, the mitochondrial acyl carrier protein, were observed in complex I is shown in pale gray, with the surface from the core subunits in the cryo-EM density for B. taurus complex I (2) and named wheat. Structural models for the supernumerary subunits are shown in color SDAP-α and SDAP-β as they are in subcomplexes Iα and Iβ, and labeled accordingly, and structural elements that cannot be assigned respectively. SDAP-α is bound to the hydrophilic domain by confidently in the current map are in blue. Subunits assignments proposed here are labeled in larger font size and underlined to differentiate them subunit B14, an LYR protein (2, 20, 21). SDAP-β is clearly vis- β from those proposed previously (2). The asterisks for PDSW and B18 indicate ible in the map from subcomplex I , on the matrix face of the that an alternative assignment in which the two assignments are exchanged membrane domain. A second LYR family complex I subunit for one another is possible. The model shown comprises the cryo-EM model (B22) is also present in subcomplex Iβ (5), raising the possibility (4UQ8.pdb) plus new elements modeled in subcomplex Iβ.

12090 | www.pnas.org/cgi/doi/10.1073/pnas.1510577112 Zhu et al. Downloaded by guest on October 2, 2021 decide which subunit is which. We tentatively propose that B18 is However, whether they retain any function in fatty-acid synthesis from chains u and v (Figs. 3B and 4) because the helices are or biosynthesis once sequestered onto the complex is unknown. more parallel than in chain t, the density more closely resembles All three CHCH domain complex I subunits (PGIV, 15 kDa, that assigned previously to the two CHCH domains in subunit and B18, human NDUFA8, NDUFS5, and NDUFB7) have now PGIV (chain X) in the cryo-EM density map from B. taurus (2), been assigned in the structure of B. taurus complex I. CHCH and the lengths of the observed helices are consistent with their domain proteins are imported to the IMS and processed by an predicted secondary structures. We do not exclude the alterna- oxidative protein folding pathway, mediated by Mia40 and Erv1, tive assignment of B18 to chain t and PDSW to chains u and v. that forms disulfides between pairs of cysteines (34). PGIV, Finally, three subunits in complex I contain double CX9C motifs: 15 kDa, and B18 are canonical substrates for Mia40, but non- PGIV was assigned previously to the heel of the complex (2), canonical substrates are also known (24) and PDSW may be B18 has been assigned here in subcomplex Iβ, and the 15-kDa folded by the same pathway. The CHCH domain family also subunit is known to be located in subcomplex Iα and on the IMS includes several respiratory-chain assembly factors and a subunit face (5, 22). On this basis, we searched for the typical two-helix of cytochrome c oxidase (24, 35). B18 is the only subunit in motif in the density maps for the B. taurus (2) and Y. lipolytica complex I that is myristoylated (36) so it is interesting that for (12) complexes and readily identified it in the expected region. another CHCH domain protein (CHCHD3) myristoylation has Consequently, we assign and model the 15-kDa subunit as a two- been proposed to be important for transporting it across the helix CHCH domain joined to helix r (Figs. 3B and 4), consistent outer membrane (37). Similarly, a CHCH domain protein (CHCHD2) with secondary structure predictions. required for the full activity of cytochrome c oxidase is trans- located to the nucleus during hypoxia to regulate transcription Discussion of an isoform of cytochrome c oxidase subunit 4 (38) suggesting Fig. 4 summarizes our current picture of the locations and that the CHCH domain subunits of complex I may similarly structures of the supernumerary subunits in B. taurus complex I. have regulatory roles, perhaps acting through the redox status The enzyme contains 45 subunits, comprising the 14 core sub- of their disulfide linkages. units, and 31 supernumerary subunits. Structural models for the In summary, we have proposed assignments for eight further core subunits, together with assignments and partial models for supernumerary subunits in the structure of mammalian complex 14 supernumerary subunits, were described in the 5-Å cryo-EM I, and thus described the most biochemically complete structural structure (2), leaving 17 subunits unassigned. Here, we have model so far. The supernumerary subunits in mammalian com- β B. taurus compared the structures of subcomplex I and intact plex I comprise approximately half of its mass, and assigning B. taurus Y. lipolytica complex I, and the density maps from the and their locations within the overall structure of the enzyme is an complexes, to propose assignments for a further eight supernu- essential step toward defining their roles, as well as a staging post merary subunits. Therefore, assignments have now been proposed on the way to a complete structural model. The supernumerary for a total of 36 subunits, and 9 subunits remain unassigned. subunit cohort in general may act as a scaffold, supporting the Little is known about three of our newly assigned subunits: core structure, or protecting it against oxidative damage. Some KFYI (human NDUFC1), the smallest mammalian subunit that supernumerary subunits may have independent functions, in occupies a peripheral location in the complex, or the B14.5b complex I assembly, mitochondrial biogenesis, or enzyme regu- (NDUFC2) or B15 (NDUFB4) subunits. NDUFC2 has been lation. Pathological mutations in the supernumerary subunits of denoted a site of accelerated replacement due to its complex I have been identified clinically in mitochondrial dis- coevolution with mitochondrial-encoded ND4 (25); this is con- eases. Our new assignments significantly advance knowledge of sistent with its assignment to a position adjacent to ND4 in our the structure of mammalian complex I, and provide new insights structure. Perhaps because it is encoded on the X into the roles of its supernumerary subunits. (26), our newly assigned ESSS subunit (human NDUFB11) has received more attention. Mutations in it cause an X-linked dis- Experimental Methods order, microphthalmia with linear skin defects syndrome (27),

Protein Preparation. Complex I was purified from B. taurus heart mitochon- BIOCHEMISTRY and two NDUFB11 isoforms have been identified, with the al- drial membranes as described previously (39) with minor modifications. − ternative form linked to apoptosis and neurodegeneration (28). Here, 150–200 mg of membranes at ∼10 mg mL 1 were solubilized using We predict that the 10-residue insertion of the alternative iso- DDM (n-dodecyl-β-D-maltoside) but the anion exchange and size-exclusion form extends the loop between the end of the TMH g and the chromatography steps were at pH 7.8 in 0.1 and 0.04% Cymal 7 (7-cyclo- start of IMS helix β5. Finally, Ser20 in a canonical motif (KRXS) hexyl-1-heptyl-β-D-maltoside), respectively, and no phospholipids were in the N-terminal matrix-domain of B. taurus ESSS is phos- added. The peak fractions from the size-exclusion column were pooled and phorylated in complex I in a cAMP-dependent manner by pro- concentrated fivefold to ∼300 μL using a 100-kDa molecular-weight cutoff tein kinase A (29). However, Ser20 is not conserved in humans Vivaspin centrifugal concentrator. Then, to remove the glycerol present in so the wider implications of this observation are unclear. the chromatography buffers, the protein was twice diluted with 5 mL of buffer containing 20 mM Tris·HCl (pH 7.8), 150 mM NaCl and 0.05% Cymal-7, Both complex I LYR proteins (subunits B14 and B22, human and reconcentrated. Finally, the protein was concentrated to 7 mg mL−1;the NDUFA6 and NDUFB9) have now been assigned in the struc- final detergent concentration was found to reach ∼2% (wt/vol) (40). ture. Of the nine further mitochondrial LYR proteins known (21), one interacts with the L-cysteine desulfurase for FeS cluster Protein Crystallization. Initial crystallization screening was performed at 22 and respiratory chain biogenesis (30), and several function as and 4 °C in MRC 96-well plates by sitting-drop vapor diffusion (200-nL respiratory chain assembly factors (21, 31). The LYR motif may complex I + 200-nL reservoir) using the Memgold HT-96 and MemStart + recruit the apparatus for FeS cluster insertion to complex II MemSys HT-96 kits (Molecular Dimensions). Crystals appeared in Memgold (mitochondrial succinate:ubiquinone oxidoreductase), and it has HT-96 [100 mM NaCl, 10 mM Hepes pH 8, and 11% PEG 1,500 (wt/vol)], but been suggested that LYR motifs in general may be used to diffracted to only ∼20-Å resolution. Extensive optimization of the conditions was then performed, including screening with the Hampton Research Ad- identify new FeS-containing proteins (32). Neither of the LYR- × × μ complex I subunits are FeS proteins; B14 is bound to the FeS- ditive Screen. The best platelike crystals of maximally 150 100 50 m appeared at 17 °C after 4 wk, and reached final size after 7–8 wk. They were containing hydrophilic arm of complex I but B22 is very distant formed by mixing 2-μL protein solution with 1.5-μL reservoir solution com- from it. Rather, the duplicated LYR–SDAP module may indicate prising 100 mM Tris-Cl (pH 7.8), 100 mM MgCl2, 100 mM NaCl, 150 mM the LYR motif recruits the SDAP protein to the complex. As the HCO2Na, 3.2 mM DDM, and 10–13% PEG 3350 (vol/vol). The crystals formed mitochondrial acyl-carrier protein, the SDAP subunits are deriv- were of subcomplex Iβ, not complex I; the subcomplex must form by frag- atized by phosphopantetheines with acyl chains attached (33). mentation of the intact complex in the crystallization solution.

Zhu et al. PNAS | September 29, 2015 | vol. 112 | no. 39 | 12091 Downloaded by guest on October 2, 2021 Data Collection and Processing. Crystals were transferred to a cryoprotectant body) and the densities from the six molecules of the asymmetric unit were

solution comprising 100 mM Tris·HCl (pH 7.8), 100 mM MgCl2, 100 mM NaCl, averaged using the NCS-averaging function in Coot (45). Subsequently, 150 mM HCO2Na, 3.2 mM DDM, and 30% PEG 3350, then flash frozen in polyalanine helices not present in 4UQ8.pdb were added to the model using liquid nitrogen. X-ray data were collected to 6.8-Å resolution from a single Coot (their orientations around the helix axis are not known), to account for crystal at 100 K at beam line ID23-1 of the European Synchrotron Radiation further clearly defined density features. The final poly-alanine model was Facility using an ADSC Q315R detector. Images were processed with XDS (41) rerefined by rigid-body refinement to Rfree = 43.3%, with all of the atomic and Mosflm (42). The crystals belong to space group P212121, with six mol- temperature factors set manually to 100. ecules (complexes) per asymmetric unit and 62% solvent content (Table S1). TMH and Secondary Structure Predictions. TMHs were predicted using TMHMM2 β Phasing, Model Building, and Refinement. The structure of subcomplex I was (46), HMMTOP2 (47), and the TOPCONS suite (48) (seven methods in total) solved by molecular replacement implemented in Phaser (43) with all of the and are presented as consensus values with less represented values in brackets atomic temperature factors set to 100. Search models comprising the bac- and single outliers discarded. Secondary structures were predicted using terial homologs of ND4 and ND5 [3RKO.pdb (10) or 4HEA.pdb (11)] failed to PSIPRED (49). provide a solution, but successful phase determination was achieved using a polyalanine search model comprising ND4 and the first 14 helices of ND5 ACKNOWLEDGMENTS. We thank Professor Randy Read (Cambridge) for from the 5-Å resolution cryo-EM structure of B. taurus complex I (4UQ8.pdb; advice on molecular replacement, the European Synchrotron Radiation ref. 2). Well-defined electron density for the supernumerary subunits was Facility (ESRF) and the Diamond Light Source for access to synchrotron revealed around the two core subunits and fitted using corresponding ele- facilities, and the staff of beam lines ID23-1, ID14-4 (ESRF) and I02, I03, I04, ments from 4UQ8.pdb. Structure refinement was performed by rigid-body and I24 (Diamond) for assistance. This work was funded by The Medical refinement using Phenix (44) (with each subunit treated as a single rigid Research Council Grants U105663141 (to J.H.) and U105184325 (to A.G.W.L.).

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