Allosteric inhibition through suppression of transient conformational states

Journal name:
Nature Chemical Biology
Volume:
9,
Pages:
462–465
Year published:
DOI:
doi:10.1038/nchembio.1250
Received
Accepted
Published online

Abstract

The ability to inhibit binding or enzymatic activity is key to preventing aberrant behaviors of proteins. Allosteric inhibition is desirable as it offers several advantages over competitive inhibition, but the mechanisms of action remain poorly understood in most cases. Here we show that allosteric inhibition can be effected by destabilizing a low-populated conformational state that serves as an on-pathway intermediate for ligand binding, without altering the protein's ground-state structure. As standard structural approaches are typically concerned with changes in the ground-state structure of proteins, the presence of such a mechanism can go easily undetected. Our data strongly argue for the routine use of NMR tools suited to detect and characterize transiently formed conformational states in allosteric systems. Structure information on such important intermediates can ultimately result in more efficient design of allosteric inhibitors.

At a glance

Figures

  1. CAP* transiently populates the active DBD state.
    Figure 1: CAP* transiently populates the active DBD state.

    (a) Relaxation dispersion profiles of 13C side chain methyls of representative CAP* DBD residues in the apo and cGMP-bound form. R2eff is the effective transverse relaxation rate, and νCPMG is the refocusing frequency of the CPMG train pulse. (b) Enhanced R2 relaxation rate (Rex) values of CAP* and CAP*–cGMP2. Rex is caused by the exchange between the ground and excited states. cGMP is shown in orange sticks. For enhanced clarity, the results are mapped on the structure of CAP with the DBD in the active conformation (CAP–cAMP2). (c) Correlation between the 13CH3 Δω and Δωdisp chemical shifts of selected DBD residues. (d) CAP* interconverts between a ground state, which adopts the inactive conformation and is 93% populated, and an excited state, which adopts the active conformation and is only ∼7% populated. cGMP binding to CAP* results in the suppression of the active conformation through an allosteric mechanism.

  2. Energy landscape of CAP* and its manipulation by the inhibitor.
    Figure 2: Energy landscape of CAP* and its manipulation by the inhibitor.

    (a) Selected region from 1H-15N HSQC spectra of CAP* in the apo form and bound to DNA. Gly173 and Gly184 are located in the recognition helix of DBD. The superscript i denotes the inactive DBD conformation, and the superscript a denotes the active conformation. The active conformation is 'invisible' because it is poorly (∼7%) populated. The chemical shifts of the active DBD conformation match with those of the wild-type CAP–cAMP2 form (depicted with gray dots)12. (b) Energy landscape of CAP* showing the two states, inactive (I) and active (A), and their fractional populations (93% and 7%, respectively). DNA binding selects the active conformation in a population shift mechanism. (c) Selected region from 1H-15N HSQC spectra of CAP* in the apo form and bound to cGMP. Binding of cGMP has no effect on the structure of DBD. (d) Selected region from 1H-15N HSQC spectra of CAP* in the cGMP- and cGMP2-DNA–bound form. DNA does not interact with CAP*–cGMP2. (e) Energy landscape of CAP* (dashed line) and CAP*–cGMP2 (orange line) showing that cGMP binding suppresses the active conformation. (f) Energy landscape of CAP*–cGMP2 showing that depletion of the active conformation in CAP*–cGMP2 results in DNA binding inhibition.

  3. Structural characterization of CAP* and CAP*–cGMP2.
    Figure 3: Structural characterization of CAP* and CAP*–cGMP2.

    (a) Structure of wild-type (WT) CAP9. The region outlined by the rectangle is used for the close-up views in the other panels. (b) Superposition of the wild-type CAP and CAP* structures. In CAP*, Leu127 and Ile128 form a hydrophobic cluster with several hydrophobic residues from CBD (Ile51, Leu61 and Trp85) thereby extending C-helices by a turn of helix. (c) Superposition of the CAP* and CAP*–cGMP2 structures. Binding of cGMP disrupts the hydrophobic cluster in CAP*, and as a result the C-helices partly unwind. (d) Superposition of the wild-type CAP and CAP*–cGMP2 structures.

  4. Allosteric inhibition by suppressing an on-pathway transiently populated intermediate.
    Figure 4: Allosteric inhibition by suppressing an on-pathway transiently populated intermediate.

    The protein interconverts between an inactive ground-state conformation (G) and an excited, active state (E). The active state is only transiently formed and is thus invisible. The ligand interacts exclusively with the excited state, giving rise to the complex (C). The inhibitor binds an allosteric site and suppresses the population of the active conformation (I), thereby resulting in binding inhibition. Structural analysis of the G and I proteins using standard approaches would reveal no structural difference in the binding site region.

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Affiliations

  1. Center for Integrative Proteomics Research, Rutgers University, Piscataway, New Jersey, USA.

    • Shiou-Ru Tzeng &
    • Charalampos G Kalodimos
  2. Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey, USA.

    • Shiou-Ru Tzeng &
    • Charalampos G Kalodimos

Contributions

S.-R.T. and C.G.K. conceived the project. S.-R.T. and C.G.K. designed the experiments. S.-R.T. performed all of the experiments. S.-R.T. and C.G.K. analyzed and interpreted the data and wrote the manuscript.

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