Structural and dynamic determinants of ligand binding and regulation of cyclin-dependent kinase 5 by pathological activator p25 and inhibitory peptide CIP

Abstract

The crystal structure of the cdk5/p25 complex has provided information on possible molecular mechanisms of the ligand binding, specificity, and regulation of the kinase. Comparative molecular dynamics simulations are reported here for physiological conditions. This study provides new insight on the mechanisms that modulate such processes, which may be exploited to control pathological activation by p25. The structural changes observed in the kinase are stabilized by a network of interactions involving highly conserved residues within the cyclin-dependent kinase (cdk) family. Collective motions of the proteins (cdk5, p25, and CIP) and their complexes are identified by principal component analysis, revealing two conformational states of the activation loop upon p25 complexation, which are absent in the uncomplexed kinase and not apparent from the crystal. Simulations of the uncomplexed inhibitor CIP show structural rearrangements and increased flexibility of the interfacial loop containing the critical residue E240, which becomes fully hydrated and available for interactions with one of several positively charged residues in the kinase. These changes provide a rationale for the observed high affinity and enhanced inhibitory action of CIP when compared to either p25 or the physiological activators of cdk5.

Figure 1

Ribbon representations of a) cdk5 from the crystal structure of the cdk5/p25 complex (chain A of 1UNL). Critical structural motifs are identified in color: activation loop (red), Gly-rich loop (green), PSSALRE sequence of interfacial helix (blue), and acidic loop L37–G43 (cyan) containing the highly charged sequence DDDDE; b) p25 from the crystal structure of the cdk5/p25 complex (chain D). Helix definitions follow Ref.; loops l₁₂ and l₃₄, both at the cdk5/p25 interface, are shown in red. The synthetic inhibitory peptide CIP results from truncation of the N- and C-terminus sequences of p25, shown in green.

Figure 2

Eigenvalues of the covariance matrix [cf. Eq.(1)] calculated from the last half of the dynamic trajectory of a) the kinase cdk5, and b) the pathological activator p25 and inhibitory peptide CIP.

Figure 3

Principal component analysis of the dynamics of cdk5: a) Collective motions of the uncomplexed kinase along the first (left) and second (right) eigenvectors. Blue and green ribbons correspond to the positions of the C_α atoms at opposite extremes of the fluctuations; red ribbons are interpolations that help visualize the range of movements. The orientations of the protein are similar to that in Figure 1; b) Same as in (a) but for cdk5 complexed with p25. The flexible segment R156–W166 of the activation loop, which defines the open and closed sub-states, is indicated by arrows. Inset: part of the activation loop containing the flexible segment R156–W166; closed sub-state from the crystal (red); open sub-state from the simulation (white); A160 and V163 side chains shown. c) Molecular surface representation of the uncomplexed (left) and complexed (right) kinase; similar orientations as in (a) and (b). Arrows point to the substrate-binding pocket, showing the enlargement induced by p25 binding. Regions lining the pocket are in red, and include the tips of the glycine-rich and acidic loops, and the flexible segment of the activation loop (cf. Fig. 1a); residues A160 and V163 are in yellow.

Figure 4

a) Hydrophobic network formed by non-polar residues in the activation loop, including I153 (yellow) during the simulation of the uncomplexed kinase; side chains of A150, G152, P154, V155 and L178 are shown in white. In the cdk5/p25 complex I153 forms instead a compact hydrophobic network with several residues in p25; b) Side-chain orientations of positively charged residues surrounding S159 in the uncomplexed kinase; side chains of R50, R125, R149, and R156 also shown. A negative charge formed by phosphorylation of S159 may perturb the reorientations of these residues and affect ligand binding.

Figure 5

Temperature factor of cdk5 and p25 from the cdk5/p25 crystal structure as reported in Ref. for chain A and D of 1UNL. Elements of secondary structure are shown at the bottom (gray: α-helix; black: β-strand). Colored segments and helix nomenclature are as in Fig. 1.

Figure 6

Ribbon representation of the uncomplexed a) p25 and b) CIP at the end of the simulation. In p25, residue M237 (green) is part of a an extended patch of non-polar residues (yellow) at the protein/water interface that keeps helix α₃ close to α₂ and α₆ through hydrophobic forces. The absence of F282 in CIP weakens this hydrophobic interaction. As a result M237 detaches from the group, and α₃ relaxes and moves away from α₂ and α₆. This change induces important restructuring of loop l₃₄ and reorientation of side chains, most notably E240, which becomes fully exposed to the solvent.

Figure 7

PCA of a) uncomplexed p25, b) uncomplexed CIP, and c) complexed p25. Ribbon colors as in Fig. 3. Only projections along the first eigenvectors are shown. White arrows indicate loops l₁₂ (right) and l₃₄ (left). Helix α₅ (at the cdk5/p25 interface) is indicated by the red arrows. Loops α_NT–α₁ (right) and α₂–α₃ (left) are at the top of the panels.

Figure 8

a) Network of non-polar residues (yellow) at the cleft between loops l₁₂ and l₃₄ (in red; cf. Figs. 1b), from the simulation of the uncomplexed p25; these residues are accessible to water and interact through hydrophobic forces. b) Hydrogen-bonding network cross-linking l₁₂ and l₃₄, from the simulation of the uncomplexed p25. c) As in (a) but for the uncomplexed CIP; the hydrophobic network is largely preserved. d) As in (b) but for the uncomplexed CIP, showing the disruption of almost all Hb between the loops, which leads to increased loops flexibility (compare Figs. 7a and 7b) and reduced cross-correlation (compare Figs. SM3 and SM4). e) Hb interactions in cdk5/p25 complex (cdk5 in white; p25 in yellow; color of critical structural motifs as in Fig.1) obtained from the simulation, showing inter-loop interaction between E240 and the backbone of D192, and intra-loop interaction between E240 and Y243, and S159 from cdk5.