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Long-range dynamic correlations regulate the catalytic activity of the bacterial tyrosine kinase Wzc

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Science Advances  18 Dec 2020:
Vol. 6, no. 51, eabd3718
DOI: 10.1126/sciadv.abd3718
  • Fig. 1 REST2 simulations on unliganded WzcCDΔC.

    (A) Cylindrical coordinate frame (defined by θ and |h|) to represent the global conformations observed in the simulations. (B) Density of states in θ-|h| space plotted using kernel density estimation; P(θ, |h|) represents the probability density. The green dot indicates the reference values for the crystal structure. Only a single major state [an open state (OS)] is sampled. (C) Close-up of the active site in the OS showing the orientation of α3 and the proximity between the Walker-A K540 and the Walker-B D642. (D) In the crystal structure [representing a closed state (CS), cyan], D642 forms a hydrogen bond with the Walker-A T541 (dotted oval). K540 (methionine in the crystal structure) is in a downward orientation similar to that in the homologous MinD (yellow). (E) In wild-type shikimate kinase (SKWT; magenta), the catalytic elements are appropriately oriented for chemistry; the Walker-A threonine (T16) is hydrogen-bonded to the Walker-B aspartate (D32) (green dotted circle). In the inactive K15M mutant (SKK15M; blue), T16 and D32 have drifted apart because of the motion of the helix (green arrow) and display orientations similar to the corresponding side chains in the WzcCDΔC OS.

  • Fig. 2 REST2 simulations on the WzcCDΔC·ATP·Mg2+ complex.

    (A) Projection onto θ-|h| space shows three distinct states: OS, CS (similar to the crystal structure; green dot), and an HS. (B) Close-up view of the active site in the OS. The catalytic elements, key ATP contacting residues, and ATP are indicated. The OS is similar to that in the apo simulations; K540 forms a salt bridge with D642; and key ATP-contacting residues (D480 and Y569) have moved away from ATP (green stick representation). In addition, the adenosine moiety of ATP is displaced from its binding pocket relative to that in the HS (tan). (C) The HS (left) shows an ordered RK-cluster; however, helices α7 and α9 of the second interaction interface (I2) are partially unfolded (green ellipses). In contrast, the OS (right) is characterized by an intact I2 but a disordered RK-cluster. (D) The two largest clusters (CS2 and CS5) obtained through an EVA-MS decomposition of the CS (also see fig. S5) are characterized by mutually exclusive distortions at the oligomerization interface: bending of α2 in CS2 (I1, red arrows) or partial unfolding of helices α7 and α9 in CS5 (I2, green ellipses).

  • Fig. 3 Active site conformation for the HS in the WzcCDΔC·ATP·Mg2+ simulations.

    (A) All catalytic and key ATP-interacting residues are shown in stick representation for an ensemble of 10 structures (left) and a single representative structure (right). Key catalytic residues on the Walker-A (dark red, K540 and T541) and Walker-B (blue, D642) motifs are indicated. ATP-coordinating residues (D480 in magenta, R490 in cyan, and Y569 in yellow), and additional ones involved in stabilizing the conformation (K492 in cyan and E572 in green) are also indicated. R490 and K492 of the RK-cluster are ordered in this state. Close-up views of the catalytic sites in the active conformations of MinD (B) and the F1-ATPase (C) are shown in approximately similar orientations as the right panel of (A). Key catalytic elements are indicated and colored similarly as their Wzc counterparts in (A).

  • Fig. 4 REST2 simulations of WzcCDΔC·ADP·Mg2+ and WzcCDΔC·ADP complexes.

    (A) θ-|h| space projection of the WzcCDΔC·ADP·Mg2+ simulations. The CS and OS are highlighted by green ellipses (left); the red dot indicates crystal structure values. Close-up (middle) of the active sites of major EVA-MS decomposed CS clusters (CS1, CS2, and CS3; also see fig. S6). ADP (cyan ellipse) and key nucleotide-coordinating residues, D480 and Y569 (green ellipse), are colored according to their constituent clusters. The right panel illustrates a representative OS structure. (B) States sampled in the WzcCDΔC·ADP simulations; the tricolor arrow indicates regions in θ-|h| space. Conformations of α2 (bending indicated by red arrows), α3, and αB of the major EVA-MS–decomposed clusters (C1, C2, and C4; also see fig. S7) are shown in the middle panel. Y569, whose position varies between the three clusters, is also indicated. Collective displacements of secondary structural elements are indicated by tricolor arrows representing specific regions in θ-|h| space. (C) Active site configurations for C1 of WzcCDΔC·ADP (left) and CS1 of WzcCDΔC·ADP·Mg2+ (right) are compared. (D) Normalized distribution of D480, Cγ-ADP, and O3′ distances (3.6 ± 0.3 Å over 16 chains in crystallo) in the WzcCDΔC·ADP·Mg2+ (blue) and WzcCDΔC·ADP (red) simulations.

  • Fig. 5 Experimental verification of in silico predictions.

    (A) Representative ITC thermograms of the interaction of WzcCDΔC with ADP (left), ADP·Mg2+ (middle), and AMPPCP·Mg2+ (right). (B) Top: Amide chemical shift perturbations (Δδ) induced by ADP on WzcCDΔC in the absence of Mg2+. Δδ values are shown only for well-resolved resonances corresponding to the apo and ADP-bound species under partial ADP saturation for the spectra that are in slow exchange. Bottom: Amide chemical shift perturbations induced by 200 molar equivalents of Mg2+ on the ADP-saturated spectra of WzcCDΔC. Red and cyan dashed lines indicate 2- and 3-SD (σ) threshold beyond the mean (<Δδ>), respectively; green bars indicate exchange broadened resonances. (C) Top: ADP-induced perturbations from the top panel of (B) depicted as red spheres on the structure of WzcCDΔC. Bottom: Mg2+-induced perturbations from the bottom panel of (B) mapped onto the structure of WzcCDΔC. Residues for which resonances show Δδ values larger than <Δδ> + 3σ or are exchange-broadened are indicated by blue and green spheres, respectively. C544, a key probe of the active site conformation and shows a large perturbation, is indicated.

  • Fig. 6 Stabilization of the CS in silico.

    (A) B-factor values from the crystal structure of WzcCD reveal a high-degree disorder at the C-terminal end of α2 (dotted circle). (B) Projection of the REST2-generated ensemble of WzcCDΔC-(G)4·ATP·Mg2+ onto θ-|h| space reveals the sampling of only a CS (the WzcCD crystal conformation is indicated by the green dot); the OS and HS seen in the WzcCDΔC·ATP·Mg2+ simulations (see Fig. 2A) are no longer seen. (C) Ten randomly selected structures superimposed on Cα atoms are shown. The α2 configuration resembles that seen in the crystal structure (i.e., no bending is observed) and the introduction of the (G)4 introduces flexibility at the C-terminal end of α2. The RK-cluster (cyan and magenta) is dynamic. However, no other structural evolution (e.g., the unfolding of the α7 or α9 helices; i.e., disruption of I2) is seen. (D) The key ATP-coordinating elements at the catalytic site are ordered, and ATP remains stably bound.

  • Fig. 7 Classical MD simulations of WzcCD·ATP·Mg2+ in the monomeric and pseudo-ring states.

    (A) θ-|h| space representation of a monomer trajectory (T3) with the points color-coded according to their time stamps (in nanoseconds; the black dot indicates crystal structure values); OS-like and CS-like states are observed. (B) Comparison of structures in the early (left; bending of α2 shown by red arrows) and late (right; displacement of Y569 and D480 from ATP indicated by blue arrows) time points from (A). (C) Conformations of the central monomer from a representative pseudo-ring trajectory (T1) plotted in θ-|h| space shows only a CS-like state. (D) The last frame from T1 shows that α2 is intact, and ATP continues to be engaged by Y569 and D480. For the monomer (E) and the central monomer in the pseudo-ring (F) simulations, the evolution of the S512,Cα-T515,Cα (proxy for α2 bending; top) and D480,Cγ-ATP,O3′ (proxy for ATP-contact; bottom) distances are shown. The left panels indicate time courses in the three independent trajectories (T1, T2, and T3), and right panels indicate frequencies of the corresponding distances. The green/black arrows highlight the inverse correlation between the distances for the monomer. Also, see fig. S8.

  • Fig. 8 Schematic representation of key conformational features identified in the REST2 simulations.

    The RK-cluster (cyan, with αB in magenta), α2 (I1, dark blue), α7 and α9 (I2, red), α3 (black), and α4 (dark red) are indicated. Monomeric, unliganded WzcCDΔC exists in the OS; binding of ATP·Mg2+ forces formation of the CS (red arrows). This state can be qualitatively conceptualized as a strained network (black arcs) of connected deformed springs. The CS is composed of several exchanging substates (clusters) with distortions that are incompatible with transphosphorylation. The most populated of these clusters show different sorts of distortions: CS5, partial unfolding of α7 and α9 (I2); CS4, enhanced disorder in the RK-cluster (dotted line) and at the N-terminal end of α2 (red arcs); and CS2, bending of α2 (I1; green arrows). Alternatively, ATP is displaced from the binding site (purple arrow) restoring the OS. These various distortions may be considered to be independent mechanisms by which this mechanical network “releases its internal strain.” A HS (proposed to represent a reactive state) that is also populated, albeit sparsely, when bound to ATP·Mg2+, is not shown for simplicity.

  • Fig. 9 Model for WzcCD activation and nucleotide exchange.

    In the monomeric state, WzcCD is stably bound to ADP in the absence of Mg2+. Although α2 is bent (green arrows), the absence of Mg2+ allows a coupled rigid body motion (red arrows; no unfolding occurs) of α7 and α9 (I2) together with α4 allowing for the proper coordination of ADP. Oligomerization and formation of the octameric ring (only three constituent monomers are represented) stabilize I1 and I2 (light green arrows) and allows formation of the CS. This state also carries some “strain” that is “released” by introducing disorder at the C terminus of α2 (red arcs), a segment that does not participate in oligomerization. In this oligomeric state, ATP·Mg2+ can be stably engaged with an affinity that is similar to (or possibly exceeds that) of ADP. Consequently, the cellular ATP/ADP ratio allows nucleotide exchange and the formation of an active conformation with ATP·Mg2+ correctly coordinated, the catalytic elements properly oriented, and the RK-cluster well ordered (thick lines) and appropriately engaged to enable catalysis.

Supplementary Materials

  • Supplementary Materials

    Long-range dynamic correlations regulate the catalytic activity of the bacterial tyrosine kinase Wzc

    Fatlum Hajredini, Andrea Piserchio, Ranajeet Ghose

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    • Environment Variability Analysis Coupled to Mean Shift (EVA-MS)
    • Figs. S1 to S9
    • References

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