Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer

Sapienza, Paul J.; Currie, Michelle M.; Lancaster, Noah M; Li, Kelin; Aubé, Jeffrey; Goldfarb, Dennis; Cloer, Erica W.; Major, Michael B.; Lee, Andrew L.

doi:10.1021/acschembio.1c00617

Cited by 7 publications

(12 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, because TrpCM has a 92% population in the low-affinity T form, the activity curve is expected to retain a sigmoidal shape if substrate binding and the associated stabilization of the R state results in a mandatory change to R in the opposite subunit. Yet as previously shown by us ( 27 ) and multiple groups ( 22 – 25 ), Trp produces a fully hyperbolic curve ( SI Appendix , Fig. S3 ).…”

Section: Resultssupporting

confidence: 80%

“…Third, apoCM does not sample R, but rather some alternate conformation. Last, in effector-free CM, we previously showed that binding a substrate analog in one protomer converts that protomer’s structure but not the unbound subunit ( 27 ), which is further proof that CM is capable of forming T:R asymmetric dimers.…”

Section: Discussionmentioning

confidence: 71%

“…1 ). Interestingly, Trp removes homotropic cooperativity between the active sites as evidenced by hyperbolic activity curves ( 22 , 24 – 27 ). X-ray crystal structures have shown that the effectors help to drive CM into T and R conformations ( 28 ).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Mixed, nonclassical behavior in a classic allosteric protein

Sapienza,

Bonin,

Jinasena

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Allostery is a major driver of biological processes requiring coordination. Thus, it is one of the most fundamental and remarkable phenomena in nature, and there is motivation to understand and manipulate it to a multitude of ends. Today, it is often described in terms of two phenomenological models proposed more than a half-century ago involving only T(tense) or R(relaxed) conformations. Here, methyl-based NMR provides extensive detail on a dynamic T to R switch in the classical dimeric allosteric protein, yeast chorismate mutase (CM), that occurs in the absence of substrate, but only with the activator bound. Switching of individual subunits is uncoupled based on direct observation of mixed TR states in the dimer. This unique finding excludes both classic models and solves the paradox of a coexisting hyperbolic binding curve and highly skewed substrate-free T–R equilibrium. Surprisingly, structures of the activator-bound and effector-free forms of CM appear the same by NMR, providing another example of the need to account for dynamic ensembles. The apo enzyme, which has a sigmoidal activity profile, is shown to switch, not to R, but to a related high-energy state. Thus, the conformational repertoire of CM does not just change as a matter of degree depending on the allosteric input, be it effector and/or substrate. Rather, the allosteric model appears to completely change in different contexts, which is only consistent with modern ensemble–based frameworks.

show abstract

Section: Resultssupporting

confidence: 80%

Section: Discussionmentioning

confidence: 71%

See 1 more Smart Citation

Mixed, nonclassical behavior in a classic allosteric protein

Sapienza,

Bonin,

Jinasena

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Verifying that this is the case would require us to make these same measurements on a sample of hTS that is bound by only a single molecule of dUMP, which is a work in progress. We have recently developed an approach that has enabled making this kind of sample of a different dimeric system ( Sapienza et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Dynamic allostery in substrate binding by human thymidylate synthase

Bonin¹,

Sapienza²,

Lee³

2022

eLife

Self Cite

View full text Add to dashboard Cite

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity—a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.

show abstract

“…Verifying that this is the case would require us to make these same measurements on a sample of hTS that is bound by only a single molecule of dUMP, which is a work in progress. Our group has recently developed an approach that has enabled us to make this kind of sample of a different dimeric system (Sapienza et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Dynamic allostery in substrate binding by human thymidylate synthase

Bonin

Sapienza

Lee

2022

Preprint

Self Cite

View full text Add to dashboard Cite

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ∼1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity—a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.

show abstract

Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer

Cited by 7 publications

References 49 publications

Mixed, nonclassical behavior in a classic allosteric protein

Mixed, nonclassical behavior in a classic allosteric protein

Dynamic allostery in substrate binding by human thymidylate synthase

Dynamic allostery in substrate binding by human thymidylate synthase

Contact Info

Product

Resources

About