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Integrating spatiotemporal features of a ligand-regulated, multi-state allosteric protein

Nature Structural & Molecular Biology volume 26, pages 816–822 (2019)Cite this article

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Abstract

Dynamic protein molecules are defined by their spatiotemporal characteristics and should thus be represented by models incoporating both characteritics. Structural biology enables determination of atomic structures of individual conformational states of a given protein. Obtaining the complementary temporal information of a given time resolution, which can be directly linked to the corresponding atomic structures, requires identifying at each time point the specific conformational state adopted by the protein. Here, we examine individual regulator of conductance to K+ (RCK) domains in the regulatory module of the MthK channel by monitoring in real time the orientation of an α-helix that is conformational-state-specific. The acquired dynamic information that specifies an RCK domain’s multi-state conformational changes, combined with already available corresponding atomic structures, enables us to establish an experiment-based spatiotemporal representation of an RCK domain, and to deduce a quantitative mechanistic model of the channel.

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Fig. 1: Structures of RCK, polarized-fluorescence intensities, dipole orientations of a fluorophore, and video frames for the tracked RCK.
Fig. 2: Observed dwell-time distributions and lifetimes of RCK conformations, and a kinetic model of RCK.
Fig. 3: Probabilities and rate constants of state-to-state transitions.
Fig. 4: Simulated kinetics and video frames for individual RCKs in the isolated regulatory module.
Fig. 5: Comparison of the kinetic model with the observed data, and simulated kinetics and video frames for a whole channel.

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Data and materials described here will be made available upon reasonable request.

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Acknowledgements

We thank Y. Zhou for technical support, Y. Jiang and R. MacKinnon for providing the complementary DNA of MthK, V. Pau and B. Rothberg for sharing their published data for comparison and P. De Weer, T. Hoshi and B. Salzberg for critiques of our manuscript at different stages of its development. This study was supported by the grant no. GM055560 from the National Institute of General Medical Sciences of the National Institutes of Health to Z.L.

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Authors and Affiliations

  1. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    John H. Lewis & Zhe Lu

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  1. John H. Lewis
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  2. Zhe Lu
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Contributions

J.H.L. and Z.L. designed the study. J.H.L. performed experiments, developed analytical tools, and analyzed the data, with the input from Z.L. J.H.L. and Z.L. interpreted the results and wrote the manuscript.

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Correspondence to Zhe Lu.

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The authors declare no competing interests.

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Peer review information: Ines Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Model for the whole MthK channel.

The gate (G) and the regulatory module (RM) both are either in an open state (o) or in a closed (c) state; RM can exist in aRM (left box) or bRM (right box). All-S2 species with or without Ca2+ bound in aRM should be excluded from the closed state of the regulatory module, but it was not explicitly done for graphic simplicity.

Supplementary Figure 2 Illustration of a single cycle of RCK’s conformational changes.

This cycle is defined by the beginning time point of one S2 event to the beginning of the next S2 event. The lifetime of S2 itself is denoted by τ2 (equation 17 in its reciprocal form). The interval between the end of the preceding S2 event and the beginning of the trailing S2 event is denoted by τ2→2 (Eq. 18), during which RCK oscillates between S1 and S3.

Supplementary Figure 3 Observed probabilities of a RCK domain to adopt the three conformational states (S1, S2 and S3) plotted against the Ca2+ concentration5.

The curves superimposed on the data were calculated using Eqs. 19–21 from lifetimes tabulated in Supplementary Table 1 and KD values in Supplementary Table 2.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3, Supplementary Tables 1–4 and Supplementary Notes 1–6.

Reporting Summary

Supplementary Video 1

Real-time video of polarized-fluorescence intensities, fluorophore dipole orientations and conformational changes of a tracked RCK domain. Intensities I0, I45, I90 and I135 (leftmost panel) recorded from the particle described in Fig. 1, their individual integrations (left-middle), angles θ, φ and Ω calculated from these intensities (right-middle) and the crystal structure of the corresponding conformational state (rightmost) are displayed point-by-point in real time and at a frame rate of 50 per second. The structures of the tracked RCK are exhibited in the form of electron density against the backdrop of the shadow of the regulatory module comprising eight RCK domains (PDB 1LNQ and 2FY8). States S1, S2 and S3 are colored yellow, blue and orange, respectively.

Supplementary Video 2

Visual illustration of the spatiotemporal model of an isolated regulatory module in the form of video played in real time and at a frame rate of 50 per second. Eight individual RCK domains independently transition among three conformations for the conditions of 0, 0.5 (near EC50) or 50 mM Ca2+ (near saturation), in accordance with temporal templates as exemplified for the Ca2+-free condition in Fig. 4a. The structures of individual conformations are exhibited in the form of a ribbon model overlaid on the space-filling model, and in a side view (left panel) and a view looking down the central axis of the regulatory module (right) (PDB 1LNQ and 2FY8). States S1, S2 and S3 are colored yellow, blue and orange, respectively. The single Ca2+ ion per RCK domain is represented by a cyan sphere.

Supplementary Video 3

Visual illustration of the spatiotemporal model of a whole channel near the EC50 of Ca2+ (1 mM) in the form of video played in real time and at a frame rate of 50 per second. The channel gate of the pore and RCK domains in the regulatory module transition among different conformations in accordance with the temporal templates shown on the left (Fig. 5d), where those rightmost points in the templates appear in real time. In the trace of the gate of the pore (top panel), the open sand closed states are colored chartreuse and maroon, respectively. The structures are simultaneously exhibited in a side view (middle panel) and a view looking down the central axis (right), in which the structure of the pore in the open state is MthK’s but that in the closed state is KcsA’s (PDB 1K4C, 2FY8 and 3RBZ). RCK’s states S1, S2 and S3 are colored yellow, blue and orange, respectively, regardless of whether the regulatory module is in configuration a or b. When all RCKs adopt S2, the pore transitions from the closed state (colored light blue) to the open state (dark blue). Ca2+ ions are presented by cyan spheres.

Supplementary Video 4

Visual illustration of the spatiotemporal model of a whole channel for three Ca2+ conditions in the form of video played in real time and at a frame rate of 50 per second. The Ca2+ concentrations are set to zero, near EC50 (1 mM) or near saturation (50 mM) in terms of the channel’s po. The structures are simultaneously displayed in a side view (left panel) and a view looking down the central axis (right), in which the structure of the pore in the open state is MthK’s but that in the closed state is KcsA’s (PDB 1K4C, 2FY8 and 3RBZ). The gate and RCK domains transition among different conformations in accordance with temporal templates, created as described for the 1 mM Ca2+ condition (Fig. 5d). RCK’s states S1, S2 and S3 are colored yellow, blue and orange, respectively, regardless of whether the regulatory module is in configuration a or b. When all RCK domains adopt S2, the pore proceeds to transition from the closed state (colored light blue) to the open state (dark blue). Ca2+ ions are presented by cyan spheres.

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Lewis, J.H., Lu, Z. Integrating spatiotemporal features of a ligand-regulated, multi-state allosteric protein. Nat Struct Mol Biol 26, 816–822 (2019). https://doi.org/10.1038/s41594-019-0276-0

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