The cause of pathogenicity of the eight disease-causing CBS mutant enzymes that have normal catalytic activity was not apparent from our initial study (12
). Purification and initial characterization of the CBS mutants expressed in the presence of various chemical chaperones showed that the inclusion of a chemical chaperone induced a specific increase of the DnaJ molecular chaperone (12
). The likely involvement of the DnaJ/DnaK/GrpE machinery resulted in an enhanced folding efficiency of the mutants thus shifting the equilibrium between the native active and misfolded inactive forms in favor of the folded fraction. However, a parallel expression of the selected mutants in the absence and presence of a chemical chaperone resulted in lower yields and lower total recovery of CBS from “non-chaperoned” samples compared to “chaperoned” ones, supporting the notion that the presence of a chemical chaperone during the mutant CBS expression mainly affects the equilibrium between folded and misfolded forms (data not shown). Additionally, our previous results suggest that the properly folded active conformations of these CBS mutants were enriched in the crude extracts (12
). Furthermore, our analyses of several CBS mutants in bacterial lysates (P49L, P78R, A114V and R125Q) revealed that their expression in the presence of various chemical chaperones did not significantly alter their proteolytic susceptibility (data not shown).
In this study, we compared conformational properties of the nine purified mutant CBS enzymes with the wild-type enzyme using far-UV CD, fluorescence and second-derivative UV spectroscopy. In the next step, we determined their structural flexibility by proteolysis with thermolysin under native conditions as well as their sensitivity towards urea-induced denaturation by pulse proteolysis. Only subtle conformational differences between the wild-type and the mutants studied were detected by the spectroscopic methods used. These findings are consistent with our previous work showing that the studied mutants did not exhibit dramatic abnormalities in the specific activities, heme saturation or native tetramer formation (12
The major differences in the properties of CBS mutants were observed when tested for their proteolytic susceptibility under native conditions. These data suggest that the mutant proteins adopted conformation, which differs from the wild-type CBS, being more flexible and exposing more hydrophobic residues for the thermolysin to attack. Moreover, the less compact structure of the mutants A114V, R125Q and E176K can also be assumed from their spectra of the second-derivative UV spectroscopy which revealed subtle decrease in the wavelength for the maxima assigned for tryptophan and tyrosine residues; similar blue shift was observed in unfolding of several model proteins (24
Interestingly, the increased structural mobility of the mutants is accompanied by impaired protein stability in urea solution only for the extensively flexible CBS mutants, R125Q and E176K, while the other CBS mutants exhibit unaffected global protein stability as was demonstrated by pulse proteolysis. The same or even higher resistance against urea-induced unfolding compared to the wild-type is also consistent with the thermostability of these mutants previously determined by absorption spectrophotometry (12
). Analogous increased proteolytic susceptibility associated with subtle conformational changes and with unaltered thermodynamic stability was reported for the yeast phosphoglycerate kinase compared to its ortholog from E. coli
). It was proposed that the discrepancies in the phosphoglycerate kinase orthologs were caused by a divergent interdomain cooperativity and consequently different mechanism of unfolding in these modular proteins. It is tempting to speculate that the increased structural mobility of majority of the CBS mutants is not caused by their low thermodynamic stability but more likely by the lower kinetic barrier of the protein unfolding; the altered interdomain communication, despite only minor conformational changes in each particular domain, may be responsible for the increased unfolding rates of these mutants.
The findings of increased proteolytic susceptibility of CBS mutants towards thermolysin are consistent with our previous study conducted directly in bacterial lysates for a different set of CBS mutants (16
). In the previous study (16
), it was proposed that higher proteolytic susceptibility of the misfolded CBS mutants in vitro
may mirror their accelerated turnover in vivo
indicating possibly an important role of proteolysis in the pathophysiology of CBS deficiency (10
). However, higher proteolytic susceptibility of all studied mutants in the previous study was associated with their increased sensitivity toward urea-induced denaturation. This discrepancy may be due to a different panel of studied CBS mutations and different degree of purity of CBS proteins in each study. In the present study, we analyzed catalytically active CBS mutants that were successfully purified to homogeneity after expression in E. coli
whereas the previous work (16
) was carried out mainly on mutants exhibiting decreased levels of catalytic activity that were not amenable to purification due to an excessive aggregation. It indicates that decreased global protein stability may be observed only for the severely affected CBS mutants but not for the mutants exhibiting subtle conformational changes. This notion is also supported by the present study showing that only the R125Q and E176K CBS mutants exhibited impaired protein stability in a urea gradient. Our present study indicates that even subtle changes in protein conformation of the catalytically active CBS mutants with normal structural stability in vitro
may lead to more rapid degradation of these variants in vivo
Since the altered response to AdoMet was proposed as one of the possible pathogenic mechanisms in CBS deficiency, particularly for the C-terminal missense CBS mutations, we compared the kinetics of proteolytic cleavage of wild-type and mutant CBS enzymes in the presence of this allosteric activator. The most rapidly cleaved CBS mutants, namely R125Q and E176K, exhibited unaltered kp
in the presence of AdoMet compared to that obtained in the absence of AdoMet. This indicates that these mutants cannot bind AdoMet and/or are locked in a specific conformation that prevents allosteric change upon AdoMet binding. This suggestion is also supported by the previously reported lack of stimulation of the catalytic activity by AdoMet and by heating of mutants (12
). More surprisingly, extremely increased proteolytic susceptibility of the A114V mutant in the presence of AdoMet suggests that its allosteric activation is likely associated with an extensive opening of the folded structure exposing naturally buried hydrophobic residues on the protein surface. Interestingly, the effect of AdoMet on native proteolysis of the R266K mutant was similar to wild type CBS but different behavior of this mutant was observed using pulse proteolysis. The presence of AdoMet led to a lower cm
value indicating that this CBS ligand decreases thermodynamic stability of the R266K mutant. This result correlates well with the previously observed decreased thermal stability and AdoMet activation of this mutant compared to wild-type CBS (17
). The impaired response to AdoMet activation was also observed for the C-terminal mutants. The proteolytic cleavage of the P422L mutant was not significantly increased and, moreover, the S466L mutant was cleaved even less rapidly in the presence of AdoMet. These findings further support the previous notion that the C-terminal mutants are locked in a specific conformation, which results in a permanently activated mutant CBS enzymes lacking proper response to AdoMet stimulation (25
). Interestingly, our results indicate that these mutant proteins may be more flexible in the absence of AdoMet than the wild-type. Even though the S466L mutant does not respond to AdoMet, it is still capable of binding it as reported previously (25
). As CBS domains in the C-terminal region very likely fold independently of the catalytic core (27
), these mutants may still bind AdoMet, but are apparently unable to rearrange their conformation. It is tempting to speculate that this locked conformation is in vivo
recognized as a misfolded structure by the cellular control machinery and are consequently targeted for degradation (28
It should be noted that the mutants P49L and P78R did not exhibit structural abnormalities by the approaches used in this study, which correlates well with their biochemical properties very similar to that of wild type enzyme (12
). The P49L exhibited high catalytic activity when expressed in the pKK expression vector without any additional tags or fusion partners (29
). On the contrary, the P78R possessed a decreased enzyme activity in the same study using the pKK construct which is consistent with a study analyzing the purified mutant reported by the Banerjee group (30
). Data on P78R indicate that pathogenicity of these mutants may be revealed by employing different expression system or using specific conditions. Nevertheless, the P49L mutation is often associated with very mild clinical manifestions (CBS Mutation Database; http://cbs.lf1.cuni.cz/cbsdata/cbsmain.htm
and Sally Stabler, oral communication at the 8th Conference on Homocysteine Metabolism, Lisboa 2011).
It should be noted that the application of a bacterial expression systems that produce the target protein with an affinity tag may have an artificial effect on quality of the purified proteins. For instance, the GST-tag that was used for the majority of the mutants may increase protein stability during the expression and subsequent purification (31
). The dramatic effect of the type and position of the employed purification affinity tag (i.e. bulky fusion partner, such as GST versus short flexible tag, such as 6xHis) and its position on the proper folding was recently demonstrated in the expression studies of the R266K mutant (17
). Nevertheless, using of affinity tags is necessary for production of target proteins in sufficient yields for their conformational analysis.
In this study, we demonstrated that protein structures of the studied CBS mutants are more locally flexible than that of the wild-type despite their normal catalytic activity and unaffected sensitivity towards urea-induced denaturation. In conclusion, the conformational analysis of the mutants using spectroscopic and proteolytic approach proved to be a useful tool for the assessment of the biochemical penalty of the CBS mutations.