An unanticipated architecture of the 750-kDa α6β6 holoenzyme of 3-methylcrotonyl-CoA carboxylase.

Abstract

3-Methylcrotonyl-CoA carboxylase (MCC), a member of the biotin-dependent carboxylase superfamily, is essential for the metabolism of leucine, and deficient mutations in this enzyme are linked to methylcrotonylglycinuria (MCG) and other serious diseases in humans. MCC has strong sequence conservation with propionyl-CoA carboxylase (PCC), and their holoenzymes are both 750-kilodalton (kDa) α(6)β(6) dodecamers. Therefore the architecture of the MCC holoenzyme is expected to be highly similar to that of PCC. Here we report the crystal structures of the Pseudomonas aeruginosa MCC (PaMCC) holoenzyme, alone and in complex with coenzyme A. Surprisingly, the structures show that the architecture and overall shape of PaMCC are markedly different when compared to PCC. The α-subunits show trimeric association in the PaMCC holoenzyme, whereas they have no contacts with each other in PCC. Moreover, the positions of the two domains in the β-subunit of PaMCC are swapped relative to those in PCC. This structural information establishes a foundation for understanding the disease-causing mutations of MCC and provides new insights into the catalytic mechanism and evolution of biotin-dependent carboxylases. The large structural differences between MCC and PCC also have general implications for the relationship between sequence conservation and structural similarity.

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An unanticipated architecture of the 750 kD holoenzyme of 3-
methylcrotonyl-CoA carboxylase
Christine S. Huang1, Peng Ge2, Z. Hong Zhou2, and Liang Tong1
1Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
2Department of Microbiology, Immunology and Molecular Genetics, California NanoSystems
Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Abstract
3-methylcrotonyl-CoA carboxylase (MCC), a member of the biotin-dependent carboxylase
superfamily, is essential for the metabolism of leucine, and deficient mutations in this enzyme are
linked to methylcrotonylglycinuria (MCG) and other serious diseases in humans 1–8. MCC has
strong sequence conservation with propionyl-CoA carboxylase (PCC), and their holoenzymes are
both 750 kD α6β6 dodecamers. Therefore the architecture of the MCC holoenzyme is expected to
be highly similar to that of PCC 9. Here we report the crystal structures of the Pseudomonas
aeruginosa MCC (PaMCC) holoenzyme, alone and in complex with coenzyme A. Surprisingly,
the structures show that the architecture and overall shape of PaMCC are strikingly different when
compared to PCC. The α subunits display trimeric association in the PaMCC holoenzyme while
they have no contacts with each other in PCC. Moreover, the positions of the two domains in the β
subunit in PaMCC are swapped relative to those in PCC. The structural information establishes a
foundation for understanding the disease-causing mutations of MCC and provides new insights
into the catalytic mechanism and evolution of biotin-dependent carboxylases. The large structural
differences between MCC and PCC also have general implications for the relationship between
sequence conservation and structural similarity.
Keywords
fatty acid metabolism; amino acid metabolism; propionic acidemia; 3-methylcrotonylglycinuria;
protein complex; biotin-dependent carboxylase; acetyl-CoA carboxylase; propionyl-CoA
carboxylase
The α and β subunits of human MCC have 42% and 34% sequence identity with those of
human PCC (Supplementary Figs. 1, 2). The α subunit contains the biotin carboxylase (BC)
and biotin carboxyl carrier protein (BCCP) domains (Fig. 1a), and a domain that mediates
BC-CT interactions (BT domain) 9. BC catalyzes the MgATP-dependent carboxylation of
biotin, and then a carboxyltransferase (CT) activity, supplied by the β subunit, catalyzes the
Correspondence and requests for materials should be addressed to L.T. (ltong@columbia.edu).
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
Author Contributions. C.S.H. carried out protein expression, purification and crystallization experiments, mutagenesis and
enzymatic assays. C.S.H. and L.T. carried out crystallographic data collection and processing, structure determination and refinement.
P.G. and Z.H.Z. carried out electron microscopy experiments. All authors commented on the manuscript. L.T. supervised the project,
analyzed the data and wrote the paper.
Author Information. The atomic coordinates have been deposited at the Protein Data Bank: 3U9R, 3U9S, and 3U9T.
The authors declare no competing financial interests.
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Published in final edited form as:
Nature. ; 481(7380): 219–223. doi:10.1038/nature10691.
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transfer of the carboxyl group to the acceptor. The β subunit contains two domains (Fig. 1a),
N and C domains, with the same backbone fold, and its active site is located at the interface
of a dimer.
Our initial interest in MCC stemmed from its distinct site of carboxylation in the substrate
(Fig. 1b). To understand the molecular basis for this activity, we produced crystals of the
MCCβ hexamer from Pseudomonas aeruginosa (PaMCCβ) that diffracted to 1.5 Å
resolution (Supplementary Table 1). This bacterial enzyme is highly homologous to human
MCC (HsMCC), with sequence identities of 47% and 65% for the α and β subunits,
respectively. To facilitate comparisons between these highly conserved enzymes, we have
numbered the residues in PaMCC according to their equivalents in HsMCC. In
Pseudomonas organisms, MCC is also involved in terpenoids metabolism 10,11.
We solved the structure of PaMCCβ using PCCβ as the model 9. However, subsequent
crystallographic analysis revealed that the positions of the N and C domains in PaMCCβ
(Fig. 1c) are swapped relative to those in PCCβ (Fig. 1d), even though the overall shapes of
the two β6 hexamers are similar. This distinct domain organization of PaMCCβ is primarily
due to a different connectivity between its N and C domains, rather than a swap of these two
domains in the primary sequence (Supplementary Fig. 3, Supplementary text). This also
leads to a large difference in the organization of the PaMCCβ dimer compared to PCCβ
(Figs. 1e, 1f). The closest structural homolog of PaMCCβ is the α subunit of glutaconyl-
CoA decarboxylase (GCDα) 12,13 (Supplementary Fig. 4). However, the sequence
conservation between PaMCCβ and GCDα (27% identity) is actually lower than that
between PaMCCβ and PCCβ (34% identity). The CoA binding sites, located in the N
domain, are swapped between MCCβ and PCCβ as well (Figs. 1e, 1f). This may be related
to the activity of MCC on the γ carbon of the substrate (Fig. 1b), as the activity of GCDα is
also on the γ carbon (Supplementary text).
Most importantly, the change in domain organization of MCCβ suggests that the overall
architecture of the MCC holoenzyme may be different as well (Supplementary text).
Therefore, we next determined the structures of PaMCC free enzyme and CoA complex at
2.9 and 3.5 Å resolution, respectively (Fig. 2a, Supplementary Table 1). Like PCC, the
holoenzyme of PaMCC contains a central β6 cylindrical core, with 3 α subunits at each end
(Fig. 2b). The overall structures of the free enzyme and CoA complex of PaMCC are
similar, although there are also recognizable differences (Supplementary Fig. 5,
Supplementary text).
Strikingly, the positions of the α subunits in PaMCC, especially their BC domains, are
entirely different from those in PCC (Fig. 2c). Rather than being splayed far apart from each
other like in PCC (Fig. 2d), the three BC domains on either end of MCC are in direct contact
with each other (Fig. 2b), burying 640 Å2 of the surface area of each BC domain. Therefore,
the BC domain shows trimeric association in the MCC holoenzyme, although this trimer is
probably unstable on its own due to the relatively small buried surface area. Overall, the
MCC holoenzyme has the shape of a cylinder, approximately 100 Å in diameter and 200 Å
tall, and this shape is remarkably different from that of PCC (Supplementary Fig. 6) 9.
As an independent verification for the crystal structure of PaMCC, we carried out electron
microscopy (EM) studies on this holoenzyme and produced a reconstruction at 12 Å
resolution (Supplementary text, Supplementary Figs. 7, 8). The crystal structure and the EM
density are in excellent agreement with each other (Figs. 2e, 2f), confirming the large
structural differences between the MCC and PCC holoenzymes.
Interactions between the α and β subunits in PaMCC are mediated predominantly by the BT
domain, and by the BCCP domain when it is in the active site of the β subunit for catalysis
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(see below). Approximately 3,000 Å2 of the surface area of each α subunit is buried in the
interface of the holoenzyme. The BT domain buries 1,500 Å2 in the interface with the β
subunit, as well as 200 Å2 in a contact with the BC domain of a neighboring α subunit (see
below). The BCCP domain in the active site of the β subunit contributes 700 Å2 to the
surface area burial, primarily through residues around the biotinylated Lys681 residue
(Supplementary Fig. 1).
The BT domain in PaMCC contains a central α-helix surrounded by a seven-stranded,
highly-twisted anti-parallel β-sheet (Fig. 3a, Supplementary Fig. 9). The overall structure of
this domain is similar to that in PCC, with an rms distance of 1.8 Å for their equivalent Cα
atoms. In addition, sequence comparisons suggest that the BT domain of HsMCC and most
other MCCs may have an eight-stranded β-barrel, which would be equivalent to that in PCC
(Fig. 3a, Supplementary text).
However, the position of the BT domain relative to the β subunit and its interactions in the
PaMCC holoenzyme are different compared to PCC (Fig. 3b, Supplementary Figs. 10, 11).
The hook of the BT domain 9, connecting the central helix to the first β-strand, is crucial for
interactions with the β subunit but has a significantly different conformation in PaMCC (Fig.
3a). The hook interacts with both the N and C domains of the β subunit in PaMCC, and
residues 542–544 in the hook form a parallel β-sheet with strand β1 (residues 95–100) in the
N domain of the β subunit (Fig. 3c, Supplementary Fig. 10, Supplementary text). Consistent
with these observations, we found that structure-based mutations in the hook could
destabilize the PaMCC holoenzyme (Supplementary Fig. 12).
In addition to the hook, there is direct contact between the BT domain of one α subunit and
the BC domain of a neighboring α subunit, where residues 604–608 (strand β27 of the BT
domain) form an anti-parallel β-sheet with strand β3 of the BC domain (Fig. 3b,
Supplementary text).
Overall, the unique interactions for the BT domain and the trimeric association of the BC
domain in the MCC holoenzyme suggest that PCC cannot form a similar architecture, and
therefore it is unlikely that the observed structural differences between MCC and PCC
represent different stages of catalysis for these enzymes.
The distance between the active sites of BC and CT is approximately 80 Å in MCC (Fig.
4a). Therefore, the entire BCCP domain must translocate during MCC catalysis, as is the
case with PCC 9 and pyruvate carboxylase (PC) 14–17. On the other hand, MCC is distinct
from PCC in that while the BT domain of an α subunit contacts its closest β subunit, its
BCCP domain is actually located in the active site of a neighboring β subunit (Fig. 4a),
another consequence of the swapping of the N and C domains in the β subunit of MCC.
We observed the binding of both CoA and BCCP-biotin to the active site of one of the β
subunits (Fig. 4a, Supplementary Fig. 13), and built a model for the bound conformation of
methylcrotonyl-CoA (Fig. 4b, Supplementary Fig. 14, Supplementary text). The binding
modes are consistent with the expected kinetic mechanism of the CT reaction 18. The N1’
atom of biotin is ~6 Å from the reactive γ carbon of methylcrotonyl-CoA in this model
(Supplementary Fig. 14). There is a large conformational change for two helices in the
active site upon CoA binding (Supplementary Fig. 15, Supplementary text).
The structure of PaMCC provides a foundation for understanding the molecular basis of its
disease-causing mutations in HsMCC, which represent one of the most frequently observed
inborn errors of metabolism 1–4,19–21. The missense mutations are distributed throughout the
holoenzyme (Fig. 4c), but their effects can be interpreted based on the structure
(Supplementary text; Supplementary Table 2). Many of the mutations are located in or near
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the BC or CT active site (for example R385S in the α subunit, A218T and V375F in the β
subunit; Fig. 4b, Supplementary Figs. 14, 16, 17). The V375F mutation may block the
binding of BCCP to the active site of the β subunit (Supplementary Fig. 17). A group of
mutations are located in or near the subunit interface in the holoenzyme, such as S535F in
the hook of the BT domain (Fig. 3c). Additional mutations are located in the hydrophobic
core of the structure, and may disrupt folding and/or stability of the enzyme.
Our structure of the PaMCC holoenzyme also has implications for the evolution of biotin-
dependent carboxylases. The structural differences between MCC and PCC suggest that
there may be two separate lineages of such enzymes that carboxylate CoA esters of organic
acids. One lineage includes PCC and acetyl-CoA carboxylase (ACC), which carboxylates
the α carbon of the acid. The other lineage targets the γ carbon of an α-β unsaturated acid
and includes MCC, GCDα, and possibly also geranyl-CoA carboxylase (GCC,
Supplementary Fig. 18) 10,11.
More importantly, the structures of MCC and PCC show that their strong sequence
conservation only ensures that the backbone folds of the domains in the two enzymes are the
same. On the other hand, the organization of these domains in the individual subunits and
especially the architecture of the subunits in the holoenzymes are remarkably different.
These observations may also have wide-ranging implications for the relationship between
sequence conservation and structural similarity in general.
Methods summary
Crystallography
The α and β subunits of PaMCC were co-expressed in E. coli, with a His-tag on the β
subunit. The PaMCC holoenzyme was purified by nickel affinity and gel filtration
chromatography. Crystals were obtained by the microbatch method under oil, and the
structures were determined by the molecular replacement method.
Electron microscopy
PaMCC sample was stained with uranyl acetate and electron micrographs were recorded at
70,000× magnification in a 200 kV electron microscope. A 12 Å resolution 3D
reconstruction was obtained from ~12,000 particle images.
Mutagenesis and kinetic studies
Site-specific mutants were designed based on the structural information, and their effects on
the formation of the holoenzyme were assessed by nickel affinity chromatography. The
catalytic activity of PaMCC was determined by a coupled enzyme assay, monitoring the
hydrolysis of ATP.
Full Methods and any associated references are available in the online version of the paper
at www.nature.com/nature.
Methods
Protein expression and purification
Full-length PaMCCβ was subcloned into the pET28a vector (Novagen). The expression
construct contained an N-terminal hexa-histidine tag, which was not removed for
crystallization. The native protein was over-expressed overnight in E. coli BL21 Rosetta
(DE3) cells (Novagen) at 20°C in the presence of 1 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) (Gold Biotechnology, Inc). The soluble protein was eluted
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from nickel affinity beads (Qiagen) and was further purified by gel filtration
chromatography with a running buffer of 25 mM Tris (pH 7.4), 250 mM NaCl, and 2 mM
DTT. The purified protein was concentrated to 20 mg/ml, supplemented with 5 % (v/v)
glycerol, flash-frozen with liquid nitrogen and then stored at −80 °C.
The PaMCC holoenzyme was over-expressed using a bi-cistronic plasmid, with PaMCCα
(untagged) placed downstream of PaMCCβ in the pET28a vector, similar to the strategy
used for the co-expression of the PCC holoenzyme 24. The holoenzyme was purified
following the same protocol as that for PaMCCβ.
Protein crystallization
Crystals were obtained with the microbatch under-oil (Paraffin oil, Hampton Research)
method at 20°C. The protein was at 20 mg/ml concentration. For PaMCCβ, the precipitant
solution contained 100 mM Tris (pH 8.5), 0.2 M (NH4)2HPO4, and 30% (v/v) PEG 200.
For the PaMCC holoenzyme, the protein was first incubated with 2 mM methylcrotonyl-
CoA for 30 min on ice. The precipitant solution contained 20% (w/v) PEG 3350 and 0.2 M
sodium malonate (pH 7.0) or 0.2 M Na2SO4. Crystals with the shape of thin plates appeared
after 10–14 days and belonged to space group P21. Most of these crystals diffracted X-rays
poorly. CoA was observed in the active site of the β subunit based on the subsequent
crystallographic analysis.
Under the same conditions, crystals with the shape of rhomboid blocks were observed after
6–8 weeks, after significant evaporation of the crystallization drops. These crystals belonged
to space group R32, and showed better X-ray diffraction. They contained the free enzyme of
PaMCC.
The crystals were flash-frozen in liquid nitrogen for diffraction analysis and data collection
at 100 K.
Data collection and structure determination
X-ray diffraction data sets were collected on an ADSC Q315 CCD at the X29A beamline of
the National Synchrontron Light Source (NSLS) at Brookhaven National Laboratory. The
diffraction images were processed using the HKL package 25. The data processing and
refinement statistics are summarized in Supplementary Table 1.
Crystals of PaMCCβ contained one β subunit in the asymmetric unit. The structure was
determined by the molecular replacement method with the program Phaser 26, using the
structure of PCCβ as the model 24. Structure refinement was carried out with the programs
CNS 27 and Refmac 28, and programs O 29 and Coot 30 were used for manual model
rebuilding. Water molecules were located automatically with the program CNS.
Crystals of the CoA complex of PaMCC holoenzyme contained one dodecamer in the
asymmetric unit. The structure was determined by the molecular replacement method with
the program Phaser, using the structure of PaMCCβ hexamer and the BC domain of
PCCα 24 as the models. One copy of the BCCP domain was also located, using the BCCP
domain of PCC as the model. However, molecular replacement calculations with the BT
domain of PCC were not successful, and the BT domain model was built based on the (omit)
electron density after structure refinement.
Crystals of the free enzyme of PaMCC contained one αβ protomer in the asymmetric unit.
The structure was determined by the molecular replacement method, using the structure of
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