A disordered encounter complex is central to the yeast Abp1p SH3 domain binding pathway

Abstract

Protein-protein interactions are involved in a wide range of cellular processes. These interactions often involve intrinsically disordered proteins (IDPs) and protein binding domains. However, the details of IDP binding pathways are hard to characterize using experimental approaches, which can rarely capture intermediate states present at low populations. SH3 domains are common protein interaction domains that typically bind proline-rich disordered segments and are involved in cell signaling, regulation, and assembly. We hypothesized, given the flexibility of SH3 binding peptides, that their binding pathways include multiple steps important for function. Molecular dynamics simulations were used to characterize the steps of binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA. Before binding, the N-terminal segment 1 of ArkA is pre-structured and adopts a polyproline II helix, while segment 2 of ArkA (C-terminal) adopts a 310 helix, but is far less structured than segment 1. As segment 2 interacts with AbpSH3, it becomes more structured, but retains flexibility even in the fully engaged state. Binding simulations reveal that ArkA enters a flexible encounter complex before forming the fully engaged bound complex. In the encounter complex, transient nonspecific hydrophobic and long-range electrostatic contacts form between ArkA and the binding surface of SH3. The encounter complex ensemble includes conformations with segment 1 in both the forward and reverse orientation, suggesting that segment 2 may play a role in stabilizing the correct binding orientation. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to the fully engaged state. In this transition, ArkA makes specific contacts with AbpSH3 and buries more hydrophobic surface. Simulating the binding between ApbSH3 and ArkA provides insight into the role of encounter complex intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs in general.

Fig 1. Description of system studied.

A) Surface view of AbpSH3 bound to ArkA from NMR [72], showing the two binding surfaces (SI (red) and SII (blue)) with bound ArkA in gray (seg1), magenta (Lys(-3)) and green (seg2). The NMR structure was determined with a longer 17-residue ArkA sequence (residues 6 through -10) [73], but only the shorter ArkA sequence is displayed. The N and C-termini are labeled. B) Sequence of ArkA used in all simulations with seg1 shown in black, the central lysine in magenta, and seg2 in green. The capping groups on the C and N-terminal ends are also shown.

Fig 2. Proposed ArkA-AbpSH3 binding model.

Two-step binding pathway with ArkA shown as a line and AbpSH3 as a circle. The heterogeneous encounter complex is proposed as an intermediate state between the unbound state and fully engaged complex. The forward and reverse rate constants for each step are labeled.

Fig 3. Characterization of unbound, bound, and encounter ensembles.

A) Conformational ensemble of unbound ArkA from REMD simulations. End-to-end distance is the distance between the C and N-terminal ends of ArkA and dihedral angle RMSD is calculated for ArkA with the lowest energy NMR structure (2RPN) as the reference [72]. Darker shading indicates a larger fraction of the total ensemble, as indicated by the color bar. B) Overlay of 13 randomly selected ArkA conformations from unbound simulations with seg1 residues Pro(2) to Pro(-2) backbone aligned. C) Overlay of 15 randomly selected ArkA conformations from bound simulations with the SH3 domain aligned. D) Overlay of 38 randomly selected ArkA conformations in the encounter complex from ArkA binding simulations with the SH3 domain aligned. AbpSH3 SI is shown in red and SII in blue. ArkA is shown in in gray (seg1), magenta (Lys(-3)) and green (seg2).

Fig 4. Quantifying bound and unbound secondary structure.

Fraction of time each ArkA residue spends in PPII Helix (A) or 3₁₀ Helix (B) during the bound and unbound simulations. The one letter codes for ArkA residues are included on the x-axis. The shaded region represents the standard deviation between independent simulations.

Fig 5. Time traces of binding and bound simulations.

Distance between ArkA and the binding surface of AbpSH3 for the first half of two example ArkA binding simulations (A, B) and one bound simulation (C). The black lines correspond to our definition of the encounter complex (23 Å) and the fully engage complex (11.5 Å). In the ArkA binding simulations, the largest box dimension is 80 Å (S3 Table) and the maximum AbpSH3 domain diameter is 33 Å. Center of mass distances between ArkA and the SH3 domain range from 11 to 64 Å in the binding simulations, while binding surface distances range from 9 to 67 Å. In the bound simulations, the binding surface distances range from 8 to 16 Å.

Fig 6. ArkA states sampled during binding and bound simulations.

Distance between ArkA and the binding surface of AbpSH3 graphed against the ArkA backbone dihedral angle RMSD for ArkA binding simulations (A) and bound simulations (B). Darker shading indicates a larger fraction of the total ensemble, as indicated by the color bar. Colored boxes partition the ensemble into four states: folded and fully engaged (blue), unfolded and fully engaged (green), folded and encounter (red), unfolded and encounter (cyan). In this figure, folded refers to the native NMR structure fold (dihedral RMSD less than 33.7°), and unfolded refers to a nonnative conformation different from the NMR fold (dihedral RMSD greater than 33.7°). Percentages indicate the occupancy of each state in the overall simulated ensemble. In the ArkA binding simulations, 8% of the ensemble is in the unbound state, which is not shown on the plot.

Fig 7. Contact maps between AbpSH3 and ArkA.

Contact maps of the fully engaged state from the bound simulations (A) and the encounter complex ensemble from the binding simulations (B). The shade of the square indicates the fraction of the ensemble with that contact. The red and blue squares along the AbpSH3 residue index show which amino acids are in SI and SII, respectively. The black line indicates the separation of seg1 and seg2, and the single letter amino acid codes are included for ArkA and the residues in SI and SII. Several contacts are formed part of the time in the encounter complex but are not occupied at all in the bound simulations, indicating that nonnative contacts are part of the encounter complex ensemble. On average in the encounter complex, seg1 is in contact with 9 SH3 domain residues, while seg2 is in contact with 6 residues. ArkA K(-3) is in contact with 2 SH3 domain residues on average in the encounter complex, consistent with its role as an important central residue for binding.

Fig 8. Forward and reverse representations of ArkA during binding.

Snapshots from MD simulation showing both the forward and reverse orientations of ArkA that are possible during binding. ArkA is shown in the stick representation with seg1 in grey, seg2 in green, and K(-3) in magenta. AbpSH3 SI is shown in red and SII in blue. The double headed arrow signifies that the ArkA orientation can flip during a simulation.

Fig 9. Long-range electrostatic interactions are non-specific in the encounter complex and specific in the bound simulations.

Frequency of particular long-range electrostatic interactions in the ArkA encounter complex (blue bars on the left) and ArkA bound simulations (orange bars on the right). The large labels indicate the ArkA residue involved in the long-range electrostatic interaction and the small labels indicate the AbpSH3 residue. Error bars represent the standard deviation between independent simulations.

Fig 10. Solvent accessibility and specific intermolecular interactions for ArkA-AbpSH3 complex.

A) Average solvent accessible surface area (SASA) of the ArkA-AbpSH3 system in the bound simulations (first bar) and binding simulations, by state of the complex. B) Occupancy of the P(2) to Y54 hydrogen bond in the bound simulations and ArkA binding simulations by state of the complex. C) Specific hydrophobic contacts between ArkA and the AbpSH3 binding surface in the fully engaged state and the encounter complex. D) Occupancy of the K(-3) to E17 salt bridge in the bound simulations and ArkA binding simulations by state of the complex. Error bars represent the standard deviation between independent simulations.