The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein

Abstract

Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability is independently regulated by the binding of S-adenosyl-methionine (SAM) to two different type of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.

Inter-domain Communication of Human Cystathionine β-Synthase

“…The T m of hCBS-FL (57.9 °C) agrees well with the value (56 °C) previously determined by differential scanning calorimetry ( 32 ). Both hCBS-FL Δ516–525 and hCBS-RD Δ516–525 exhibited a lower T m (49.2 and 33.5 °C) than hCBS-FL, likely an impact of removing loop 516–525 and the catalytic domain on the remainder of the polypeptide.…”

“…Second, the previously reported isothermal titration calorimetry data may be alternatively interpreted ( e.g. negative cooperativity ( 32 , 37 ), where AdoMet binding to the first S2 site decreases its affinity to other S2 sites present in the hCBS oligomer, such that stoichiometry may depend on AdoMet concentration). Further work is warranted to answer this ambiguity.…”

Oligomeric status of human cystathionine beta‐synthase modulates AdoMet binding

“…Figure 2 shows representative raw titrations and binding isotherms, while Table 1 summarizes thermodynamic parameters for AdoMet binding as determined by ITC. As previously reported [6,9,14], AdoMet binding to CBS WT is consistent with a binding capacity of about 1.5 moles of AdoMet/monomer into two types of independent binding sites. Attempts to analyze these binding isotherms using a sequential binding model with a total binding capacity of four binding sites per tetramer did not provide convergent fittings (data not shown), supporting the notion that these isotherms cannot be satisfactorily explained by four dependent (i.e.…”

“…Figure shows representative thermal denaturation profiles, while Table summarizes key denaturation parameters for the regulatory (RD) and the catalytic domains (CD) obtained for CBS WT and CBSΔ516‐525 variants using a two‐state irreversible denaturation model . As previously reported , thermal denaturation profile of CBS WT showed two main transitions with T m values of 51.2 °C and 67.2 °C corresponding to denaturation of RD and CD, respectively. The CBSΔ516‐525 variants presented similarly well resolved two thermal transitions to CBS WT, which T m for both RD and CD denaturation were generally up‐shifted by 2 °C.…”

“…Figure 3 shows representative thermal denaturation profiles, while Table 2 summarizes key denaturation parameters for the regulatory (RD) and the catalytic domains (CD) obtained for CBS WT and CBSD516-525 variants using a two-state irreversible denaturation model [6]. As previously reported [6,9,14], thermal denaturation profile of CBS WT showed two main transitions with T m values of 51.2°C and 67.2°C corresponding to Fig. 3.…”