Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling

Abstract

Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by S-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms.

Conformational Properties of Nine Purified Cystathionine β-Synthase Mutants

“…The presence of AdoMet increased the stability of wild-type CBS as reported previously (18,23) whereas the c m values for the majority of mutants were not changed (Tab. 2).…”

“…1). We have demonstrated previously that allosteric activation of CBS is associated with opening of the protein conformation and that the wild-type CBS is cleaved in the presence of AdoMet more rapidly with doubling of the kinetic constant (18). The similar increase in cleavage rate was also observed in this study for all AdoMet-responsive CBS mutants, namely the P49L, P78R, A114V and R266K.…”

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

“…A major contribution of our structure is the unveiling of the relative orientations of the regulatory and catalytic domains in hCBS, which are in striking contrast to the orientation of both the previous in silico models (24,25) and the dCBS structure (21). The hCBS forms a basket-shaped symmetrical dimer in which the catalytic core of each subunit interacts with both the catalytic core and the regulatory domain of the complementary subunit (Fig.…”

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S -adenosylmethionine

“…More than a decade ago, we and others hypothesized that hCBS might exist in two different conformations: a "basal" state with low activity, where the C-terminal regulatory domain would restrict the access of substrates into the catalytic site, and an AdoMet-bound "activated" state, where the AdoMet-induced conformational change would allow for enzyme activation (16,19). Recently, we have unveiled the relative orientations of the regulatory and catalytic domains in hCBS (18), which were in a striking contrast to those of both the previous in silico models (20,21) and the Drosophila melanogaster (dCBS) structure (22). Our data showed that, although the pairing mode and the orientation of catalytic cores are similar in both insect dCBS and hCBS, the position of their regulatory domains is markedly different (18).…”