Structural basis of regulation and oligomerization of human cystathionine β-synthase, the central enzyme of transsulfuration

Abstract

Cystathionine β-synthase (CBS) controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H2S. CBS condenses serine and homocysteine to cystathionine with the help of three cofactors, heme, pyridoxal-5'-phosphate, and S-adenosyl-l-methionine. Inherited deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur metabolism. We present the structure of the human enzyme, discuss the unique arrangement of the CBS domains in the C-terminal region, and propose how they interact with the catalytic core of the complementary subunit to regulate access to the catalytic site. This arrangement clearly contrasts with other proteins containing the CBS domain including the recent Drosophila melanogaster CBS structure. The absence of large conformational changes and the crystal structure of the partially activated pathogenic D444N mutant suggest that the rotation of CBS motifs and relaxation of loops delineating the entrance to the catalytic site represent the most likely molecular mechanism of CBS activation by S-adenosyl-l-methionine. Moreover, our data suggest how tetramers, the native quaternary structure of the mammalian CBS enzymes, are formed. Because of its central role in transsulfuration, redox status, and H2S biogenesis, CBS represents a very attractive therapeutic target. The availability of the structure will help us understand the pathogenicity of the numerous missense mutations causing inherited homocystinuria and will allow the rational design of compounds modulating CBS activity.

Fig. 1.

Architecture and biochemical properties of hCBSOPTΔ516–525. (A) In hCBSOPTΔ516–525, unlike the truncated 45-kDa hCBS lacking the Bateman module (red), the distribution of the functional domains is not affected as compared with the native hCBS. (B and C) SDS/PAGE (B) and native PAGE (C) Western blots of the purified enzymes probed with monoclonal anti-CBS antibody show the predominantly dimeric form of hCBSOPTΔ516–525 in contrast to native tetramers of wild-type hCBS and dimers of 45-kDa hCBS. (D) CBS activities ± 300 µM AdoMet show that our hCBSOPTΔ516–525 construct has basal activity and is activated by AdoMet to a similar extent as wild-type hCBS, unlike the constitutively activated 45-kDa hCBS.

Fig. 2.

Topology and structure of hCBSOPTΔ516–525. (A) Cartoon presentation of the secondary structures within the C-hCBSOPTΔ516–525 amino acid sequence. (B) Structural superimposition of the truncated 45-kDa hCBS protein (green; PDB ID code 1JBQ) with the catalytic core of the full-length hCBS protein (blue). The loops L145–148, L171–174, and L191–202 are located at the entrance of the PLP cavity and regulate the access of substrates into the catalytic site. As depicted, the loop L191–202 is disordered in the truncated 45-kDa hCBS protein and thus is not visible. The open conformation adopted by the loops L145–148 and L171–174 in the absence of the regulatory domain explain why the truncated enzyme is activated. (C) Interface between the catalytic core (blue) and the Bateman module (red) in the basal form of the full-length enzyme. The truncated 45-kDa hCBS protein (green) is superimposed for comparison. As depicted, the Bateman module pushes the entrance loops toward the PLP cavity, thus hindering the access of substrates. (D) The 3D structure of the Bateman module, which contains two tandem-repeated CBS motifs (CBS1, residues 412–471, and CBS2, residues 477–551). Secondary structure elements have been colored as in A. S1 and S2 designate the proposed AdoMet-binding sites.

Fig. 3.

Structural differences between the basal hCBS and the constitutively activated dCBS. (A and B) hCBS (A) and dCBS (B) monomers comprise an N-terminal catalytic core (dark blue) connected via a linker (red) with the C-terminal regulatory domain (yellow). (C and D) The structures of dimeric hCBS (C) and dCBS (D) show the strikingly different orientation of the regulatory domains toward the catalytic cores. The polypeptides within the dimers are shown in orange and blue. (E and F) Top views on the regulatory domains in hCBS (E) and dCBS (F) highlight their distinct arrangement. The Bateman modules of the two subunits in the dCBS dimer form a tight disk-shaped CBS module in contrast to hCBS, in which the regulatory domains are far apart and do not interact with each other.

Fig. 4.

Structural elements determining access to the active site. (A) Surface representation of the 45-kDa hCBS shows that, in the absence of the regulatory domain, the catalytic cavity remains open and the PLP is exposed. (B) In contrast, in the basal form of hCBS, the entrance to the cavity is occluded by the Bateman module by the closed conformation of the loops L145–148, L171–174, and L191–202. (C) A zoom-in view of the interface region between the catalytic core and Bateman module showing the structural elements at the entrance of the catalytic site in hCBS (red) and in the 45-kDa hCBS (gray). (D) Superimposition of the region shown in C for the basal form of hCBS (cyan), for the pathogenic D444N hCBS mutant (red), and for dCBS in the absence (yellow) or the presence (green and gray, respectively) of bound substrates.

Fig. 5.

Putative AdoMet-binding sites in hCBS. (A) Site S1. The entrance of S1 is sterically hindered by the presence of structural elements from the catalytic core (cyan) of a complementary monomer in the dimer. Additionally, bulky hydrophobic residues (Y484, H507, and F508) occupy the cavity and probably impede the binding of AdoMet at this site in the basal form. Residue N463, instead of the conserved aspartate that would stabilize the nucleotide ribose ring, could play a similar role. (B) Site S2. In contrast, the cavity of S2 is solvent exposed and is not blocked by bulky residues. The site S2 shows features similar to the AdoMet-binding protein MJ0100 (27): a hydrophobic cage to host the adenine ring of AdoMet, conserved aspartate (D538) and threonine (T535) residues to stabilize the ribose ring, and a hydrophobic residue (I537) preceding D538 to accommodate the alkyl chain of AdoMet. Noteworthy are the residues linked with pathogenic mutations (identified in red).

Fig. 6.

The proposed model of hCBS activation. (A and B) Ribbon (A) and surface (B) representations of a model of the activated form of hCBS upon binding of AdoMet at site S2. The loops controlling the access to the PLP cavity (a: L191–202; b: L171–174; c: L145–148) are open, thus allowing the access of substrates. (C and D) Ribbon (C) and surface (D) representations of the basal form of hCBS obtained from the crystals. The loops controlling access to the PLP cavity are closed and occlude the entrance of substrates into the catalytic cavity.

Fig. 7.

The proposed model of the hCBS tetramer. The hCBS tetramerization is sustained by the interactions of each Bateman module with the Bateman module and with the catalytic cores of the complementary dimer. The tetramer is stabilized by interactions between loop 513–529, which serves as a hook locking the two dimers together, and the residues located at the cavity formed by the helices α6, α12, α15, and α16.