Conformational Properties of Nine Purified Cystathionine β-Synthase Mutants

Abstract

Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine beta-synthase (CBS) deficiency. In our previous studies, we have successfully expressed, purified and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence and second derivative-UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to the wild-type although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible towards cleavage suggesting their increased local flexibility or propensity to local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the cleavage rate of wild-type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of wild-type and the other mutants. Taken together, the proteolytic data show that the conformation of pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are a useful tool for the assessment of the biochemical penalty of missense mutations in CBS.

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

“…S1 and S2). These findings are in agreement with the recent biochemical, spectroscopic, and calorimetric studies demonstrating that AdoMet alleviates the intrasteric block imposed by the regulatory domain without significant conformational rearrangements (19,31). Instead, our data strongly suggest that the active conformation of hCBS differs significantly from that found in dCBS (21).…”

“…With the aim of unraveling the AdoMet-mediated allosteric regulation of hCBS activity, we further tried to obtain crystals of the hCBS-AdoMet complex. Because of the controversy found in the literature about whether (28)(29)(30) or not (19,31) AdoMet induces a large conformational change upon binding to hCBS and to avoid possible structural artifacts caused by preexisting crystal packing, we first performed cocrystallization experiments of hCBS with AdoMet instead of soaking previously grown crystals in solutions containing the allosteric regulator. To assure complete occupancy of all potential binding sites, an excess of AdoMet (molar ratio 10:1) was added to the protein solution subjected to crystallization.…”

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

“…We recently demonstrated that the structure of D444N, a pathogenic mutant with increased basal activity and impaired response to AdoMet (25,32), closely resembles that of basal hCBS, showing only small structural rearrangements limited to a slight displacement of helices α18, α19, and α22 within the regulatory domain (18). This arrangement, concordant with recent findings indicating that activation of hCBS by AdoMet elapses without significant alteration of secondary structure elements (26,27), seemed to contradict former data claiming the occurrence of large conformational rearrangements to reach the activated conformation (16,19,25). Nevertheless, the effect induced by the D444N mutation allowed us to explain higher basal activity of this mutant, in which the loops at the entrance of the catalytic site could find sufficient space to move freely (18).…”

“…These findings are in agreement with the much higher basal activity of dCBS and its inability to bind or to be regulated by AdoMet (23,24) and suggest that the structural basis underlying the regulation of the human enzyme markedly differs from CBS regulation in insects or yeast (24). Taken together, the available data indicate that binding of AdoMet to the Bateman module weakens the interaction between the regulatory domain and the catalytic core although the mechanism and the magnitude of the underlying structural effect are still under debate (16,19,(25)(26)(27).…”

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

“…More recently, we have determined the crystal structure of an optimized dimeric full-length hCBS construct lacking 10 residues in the regulatory domains [3], that provides a structural framework to explain SAM mediated regulation of hCBS function and stability. However, comprehensive structural-energetic studies on major forces contributing to hCBS stability as well the impact of disease-causing mutations on the thermodynamic and kinetic stability of hCBS are scarce [4, 6, 7]. …”