Table 1.
Reported disease-causing mutations of human HAD gene.
Figure 1.
Mapping disease related mutations into human HAD crystal structure (PDB entry 1F0Y).
Two monomers of HAD are colored in blue and grey, respectively. The carbon atoms of NAD+, acetoacetyl-CoA (AACoA), side chains of mutated residues and catalytic His-Glu pair of one monomer (blue) are colored in green, yellow, magentas and salmon, respectively. Regions colored in lemon refer to the in-frame deletion from 171–200 (c.547-3_549 deletion in HAD gene). If not mentioned, all the structure illustrations were generated with PyMOL (http://www.pymol.org. Accessed 2014 March 26th.).
Table 2.
Data collection and refinement statistics*.
Table 3.
Kinetic parameters of wild type and mutant cHAD enzymes.
Figure 2.
Overall structure of cHAD and protein primary structure comparison between cHAD and its homologues.
(A) Ribbon diagram of cHAD crystal structure (PDB entry 4J0F, this work) colored in purple. The loop between helix α2 and α3 with weak electron density is indicated by dotted line. (B) Tertiary structure alignment among 3-hydroxyacyl-CoA dehydrogenases from C. elegans (purple, PDB entry 4J0F), human (blue, 3HAD) and E. coli (yellow, 3MOG). The region from residue 286 to 475 in E. coli HAD is not shown. (C) Sequence alignment among 3-hydroxyacyl-CoA dehydrogenases from Caenorhabditis elegans (C. elegans, GenBank accession No. CAA80153.1), Homo sapiens (human, GenBank accession No. CAA65528.1), Sus scrofa (pig, GenBank accession No. AAD20939.1), Mus musculus (mouse, GenBank accession No. BAA06122) and Escherichia coli (E. coli, GenBank accession No.NP_415913.1, residues 1–286). The transit peptide sequence was excluded from human, pig and mouse HAD sequences. The secondary structures are corresponding to cHAD. The catalytic His-Glu pair is boxed in black. The disease related point mutations in Table 1 are boxed with dashed line. The conserved glycine residues flanking C-terminal domain helices are indicated by triangles. The conserved Arg and Glu forming salt-bridge on dimerization interface are indicated by asterisks; and the conserved hydrophobic residues on the dimerization interface are indicated by “#”. The labels of secondary structure are corresponding to those in (A).
Figure 3.
Dimerization interface of cHAD.
(A) Ribbon (left) and surface (right) diagram of the crystal structure of cHAD dimer in an asymmetric unit. The two molecules, subunit A and subunit B, colored in cyan and yellow respectively in the ribbon diagram, are arranged in a “tail-to-tail” manner through interactions between their C-terminal domains. The loop between helix α2 and α3 with weak electron density is indicated by dotted line. In the surface diagram, only secondary structures involving dimerization are depicted. (B) An “open-book” view of the dimerization interface between subunit A and B. The negatively charged, positively charged, polar, hydrophobic and glycine residues on the surface are represented in red, blue, green, yellow and white, respectively. The contact sites of the dimerization interface via α8/α8′, α9/β7′–β8′ and α9′/β7–β8 are indicated by the red dotted ellipse, green dotted rectangle and blue dotted rectangle. (C) Core dimerization interface. A combined ribbon and stick model illustrates both electrostatic (left) and hydrophobic (right) interactions between each α8 helix of two subunits. Salt bridges are indicated with the dashed lines. (D) Gel filtration profile of the wild type and mutated cHADs. Equal amount of protein (100 µg) was injected onto a pre-equilibrated Superdex 200 column (10/300 GL; GE healthcare) and eluted at a flow rate of 0.5 ml/min.
Figure 4.
Negative cooperation effect of NADH binding within the cHAD dimer.
(A) A combined surface and stick model presents the distances between the side chain of intrinsic W260 and bound NADHs of subunit A (cyan) and subunit B (yellow). The positions of NADHs are determined by superposition of the crystal structure of human HAD·NADH complex (PDB entry 1F17) with the present cHAD crystal structure. (B) Fluorescence resonance energy transfer (FRET) spectrum by titrating NADH with the wild type cHAD. The excitation wavelength is 270 nm and the emission wavelength is scanned from 290 to 500 nm. The FRET signal appears at the wavelength of 460 nm. (C) Hyperbolic curve of the FRET fluorescence signal at 460 nm against the titration of NADH concentration for the wild type cHAD. The insert represents the logarithmic Hill plot between 10 and 90% active site saturation.
Figure 5.
Characterization of the charge transfer complex intermediate formation.
(A) A combined surface and stick model presents a structural model of cHAD ternary complex with the NAD+ and AACoA bound. The α8 helices of the dimer are depicted as ribbons. The positions of NAD+ and AACoA are determined by superposition of the structure of human HAD·NAD·AACoA complex (PDB entry 1F0Y) with the present cHAD crystal structure. (B) Difference absorption spectra of the cHAD·NAD+·AACoA complexes for the wild type (purple), R204A mutant (green), Y209A mutant (blue) and R204A/Y209A mutant (red) at a ligand concentration of 2 mM as described in MATERIALS AND METHODS. (C) Effects of mutations on charge transfer complex formation. The columns filled in white represent the net absorbance of ternary complex formed by cHAD and its variants at 412 nm (scaled by left vertical axis), while the columns filled in black represent their Vmax values determined in kinetic experiments (scaled by right vertical axis). (D) Thermo shift assay of cHAD and its variants. (E) The critical melting temperature (Tm) of cHAD and its variants from the thermo shift assay in (D). (F) The root-mean-square fluctuations (RMSF) of cHAD (wild type at left, R204A at middle and R204A/Y209A at right) N-terminal domain residues (1–198) for each subunit (chain 1 and 2). The significant fluctuation regions (R1, a.a. 60–80) are label accordingly. (G) The RMSFs of cHAD (wild type at left, R204A at middle and R204A/Y209A at right) C-terminal domain residues (199–297) for each subunit (chain 1 and 2). The significant fluctuation regions (R2, a.a. 260–280) are label accordingly. (H) Cartoon representation of the crystal structure of cHAD with one subunit colored in cyan and another in gold. The significant fluctuation regions R1 (a.a. 60–80) and R2 (a.a. 260–280) observed in molecular dynamics simulation (F and G) are colored in red and blue, respectively.
Table 4.
Averaged Cα distances of corresponding residues from molecular dynamics simulationsa.