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 3 10 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.
Protein-protein interactions are involved in a wide range of cell 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 that cannot always capture low populations of intermediate states. 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 binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA, to capture all steps along the pathway. 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 3 10 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 revealed 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 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 3 intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs more generally. Author SummaryComplex cellular processes are mediated by interactions between proteins, and to determine how these physical interactions affect cellular function we need to understand the protein binding pathway. Many protein interaction domains, such as the SH3 domain, bind to intrinsically disordered proteins in a coupled folding and binding process. Using molecular dynamics simulations, we find that the binding of the disordered ArkA peptide to the yeast Abp1p SH3 domain proceeds through a flexible, disordered encounter complex before reaching a more stable fully bound state. This encounter complex is stabilized by nonspecific long-range electrostatic interactions and nonspecific hydrophobic interactions between the peptide and domain. Our simulations highlight the important role of hydrophobic interactions in the entire SH3 binding process, both nonspecific hydrophobic contacts in the encounter complex and specific hydrophobic contacts in the fully bound complex. The enc...
characterize the complexes of Hsp70 and TMR-amylin. Radius of gyration of the complexes were below the detection limit of MALS. Molecular weight measured by MALS, and Hydrodynamic size and fluorescence brightness of the complexes measured by FCS indicate interactions of Hsp70 with monomeric or small oligomeric forms of amylin. Taken together our results indicate that Hsp70 inhibits aggregation of amylin possibly by preventing primary nucleation by binding to the 'misfolded' monomers and/or small oligomers of amylin. 405-Pos Board B175Conformational Changes and Flexibility for ArkA Binding to Abp1-SH3 SH3 domains are the most common protein interaction domains and are found across all forms of life. However, little is known about how flexible peptides bind to these domains. These flexible peptides are often intrinsically disordered proteins (IDPs) which are difficult to model using only experimental methods. One SH3 domain found in yeast, Abp1SH3, has a binding site for the ArkA IDP. Molecular dynamics simulations were used to model the Abp1SH3 domain and the Abp1SH3-ArkA complex. SH3 domains are believed to have a two-step binding process in which part of the peptide binds to Surface I (SI) and afterwards, the rest of the protein binds to Surface II (SII). It was found that there is a greater amount of flexibility in the residues located in SI and SII compared to other residues in the SH3 domain. Also, several side chain dihedral angles in SI and SII have different angle preferences between the free and bound states. Mutations to the ArkA peptide were made including several residues mutated to alanine and a K-3R mutation. It was found that although lysine and arginine are similar in structure, this mutation resulted in fewer contacts between ArkA and Abp1.The mutations to alanine gave insight to the specificity SII has for binding the ArkA peptide, shown by a change in binding pattern between the proteins. Simulations were also run with a linker between Abp1 and ArkA, to mimic experimental procedures. It was found that there are similar contacts between Abp1 and ArkA with and without the linker, however, there is an increased flexibility in Abp1 in the presence of the linker compared to the normal bound structure. This increased flexibility may be compensating for the reduction of entropy caused by the linker.
SH3 domains are the most common protein interaction domains and are found across all forms of life. However, little is known about how flexible peptides bind to these domains. These flexible peptides are often intrinsically disordered proteins (IDPs) which are difficult to model using only experimental methods. One SH3 domain found in yeast, Abp1SH3, has a binding site for the ArkA IDP. Molecular dynamics simulations were used to model the Abp1SH3 domain and the Abp1SH3-ArkA complex. SH3 domains are believed to have a two-step binding process in which part of the peptide binds to Surface I (SI) and afterwards, the rest of the protein binds to Surface II (SII). It was found that there is a greater amount of flexibility in the residues located in SI and SII compared to other residues in the SH3 domain. Also, several side chain dihedral angles in SI and SII have different angle preferences between the free and bound states. Mutations to the ArkA peptide were made including several residues mutated to alanine and a K-3R mutation. It was found that although lysine and arginine are similar in structure, this mutation resulted in fewer contacts between ArkA and Abp1.The mutations to alanine gave insight to the specificity SII has for binding the ArkA peptide, shown by a change in binding pattern between the proteins. Simulations were also run with a linker between Abp1 and ArkA, to mimic experimental procedures. It was found that there are similar contacts between Abp1 and ArkA with and without the linker, however, there is an increased flexibility in Abp1 in the presence of the linker compared to the normal bound structure. This increased flexibility may be compensating for the reduction of entropy caused by the linker.
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