Tetratricopeptide Repeat Motif-mediated Hsc70-mSTI1 Interaction

The Hsp-organizing protein (HOP) binds to the C termini of the chaperones Hsp70 and Hsp90, thus bringing them together so that substrate proteins can be passed from Hsp70 to Hsp90. Because Hsp90 is essential for the correct folding and maturation of many oncogenic proteins, it has become a significant target for anti-cancer drug design. HOP binds to Hsp70 and Hsp90 via two independent tetratricopeptide (TPR) domains, TPR1 and TPR2A, respectively. We have analyzed ligand binding using Poisson-Boltzmann continuum electrostatic calculations, free energy perturbation, molecular dynamics simulations, and site-directed mutagenesis to delineate the contribution of different interactions to the affinity and specificity of the TPR-peptide interactions. We found that continuum electrostatic calculations could be used to guide protein design by removing unfavorable interactions to increase binding affinity, with an 80-fold increase in affinity for TPR2A. Contributions at buried charged residues, however, were better predicted by free energy perturbation calculations. We suggest using a combination of the two approaches for increasing the accuracy of results, with free energy perturbation calculations used only at selected buried residues of the ligand binding pocket. Finally we present the crystal structure of TPR2A in complex with its non-cognate Hsp70 ligand, which provides insight on the origins of specificity in TPR domain-peptide recognition.The Hsp-organizing protein (HOP) 4 plays a key role in in vivo folding by bringing the chaperones Hsp70 (or its constitutively expressed isoform, Hsp70) and Hsp90 together into a complex (1) (see Fig. 1), where specific substrate proteins are passed over from Hsp70 to Hsp90. Partially folded Hsp90 client proteins are transferred from Hsp70 to Hsp90, where the final steps of folding are completed (2, 3). While bound to the Hsp90, the client protein then becomes functional, e.g. hormone receptors can bind their ligands, and then are released. Both chaperones must be brought together by their interaction with HOP to accomplish this (see Fig. 1). The whole Hsp90 functional cycle involves also other co-chaperones, such as p23, other TPR proteins, and ATP binding and hydrolysis, and is fairly complicated and incompletely understood (4). However, it is clear that HOP facilitates the interaction of Hsp70 and Hsp90 and thus is important in providing the link between the chaperones in the early part of Hps90 functional cycle. Hsp90 client proteins include nuclear hormone receptors, p53, and other transcription factors and various kinases, such as Atk and Her2. Many of these are oncogenes, making Hsp90 a significant anti-cancer drug target (5). It is therefore of considerable importance to better understand the chaperone cycle of Hsp90 and in particular its interaction with essential co-chaperones, such as HOP. The interactions of Hsp70 and Hsp90 with HOP also provide an excellent model system in which to study the contribution of electrostatics to protein-peptide interactions, as will bec...

Section: Discussionmentioning

confidence: 99%

Electrostatic Interactions of Hsp-organizing Protein Tetratricopeptide Domains with Hsp70 and Hsp90

Kajander

Sachs

Goldman

et al. 2009

“…Hsc70 has been shown to play a critical role in the processing of voltage-gated K ϩ channels involved in the trafficking of axonal protein vesicles and the delivery of these channel-containing vesicles to the cell surface (59). Interestingly, recent studies on the binding domains within the Hsp70 and Hsp90 proteins have shown that there may be charge interactions between the various cargo proteins and chaperones (60,61), suggesting a possible role for the charged lysine residue at position 197 of hIK1 in chaperone-mediate protein trafficking.…”

Section: Discussionmentioning

confidence: 99%

Role of an S4-S5 Linker Lysine in the Trafficking of the Ca2+-activated K+ Channels IK1 and SK3

Jones

Hamilton

Devor

2005

We have investigated the role of the S4-S5 linker in the trafficking of the intermediate (human (h) IK1) and small (rat SK3) conductance K ؉ channels using a combination of patch-clamp, protein biochemical, and immunofluorescence-based techniques. We demonstrate that a lysine residue (Lys 197 ) located on the intracellular loop between the S4 and S5 domains is necessary for the correct trafficking of hIK1 to the plasma membrane. Mutation of this residue to either alanine or methionine precluded trafficking of the channel to the membrane, whereas the charge-conserving arginine mutation had no effect on channel localization or function. Immunofluorescence localization demonstrated that the K197A mutation resulted in a channel that was primarily retained in the endoplasmic reticulum, and this could not be rescued by incubation at 27°C. Furthermore, immunoblot analysis revealed that the K197A mutation was overexpressed compared with wild-type hIK1 and that this was due to a greatly diminished rate of channel degradation. Co-immunoprecipitation studies demonstrated that the K197A mutation did not preclude multimer formation. Indeed, the K197A mutation dramatically suppressed expression of wild-type hIK1 at the cell surface. Finally, mutation of this conserved lysine in rat SK3 similarly resulted in a channel that failed to correctly traffic to the plasma membrane. These results are the first to demonstrate a critical role for the S4-S5 linker in the trafficking and/or function of IK and SK channels.The human (h) 3 intermediate conductance Ca 2ϩ -activated K ϩ channel IK1 is a member of the KCNN gene family, which also includes SK1-3, the small conductance Ca 2ϩ -activated K ϩ channels. Within this channel family, there is ϳ40% amino acid sequence homology, with the greatest level of identity occurring in the pore and proximal C terminus, a region known to constitutively bind calmodulin (1, 2), thereby conferring Ca 2ϩ sensitivity to these channels. The hIK1 channel is now known to be the Gardos channel involved in red blood cell volume regulation (3). hIK1 is also known to be involved in the Ca 2ϩ -dependent regulation of Cl Ϫ secretion across intestinal and airway epithelia (4 -8). More recent work has demonstrated that the progression of breast cancer cells through the cell cycle is dependent on membrane hyperpolarization resulting from the activation of hIK1 channels (9). hIK1 has also been shown to play a role in both macrophage activation and T-lymphocyte proliferation (10 -12). The critical role that pharmacological modulation of hIK1 may play has been revealed by the demonstration that blockers of hIK1 reduce the autoimmune response to experimental encephalomyelitis in mice (13), reduce brain edema following traumatic brain injury (14), and prevent restenosis following balloon angioplasty (15). Also, openers have been shown to alter vascular tone and hence may modulate peripheral blood pressure (16,17). A great deal of research has focused on both the biophysical and pharmacological properties of hIK1 (18 -20)...

“…Initially, we identified Hop as a novel target for S100A2 and S100A6. Hop (11,33,34). Therefore, we speculated that these S100 proteins are able to compete with Hsp binding to Hop in intact cells.…”

Section: Discussionmentioning

confidence: 99%

Interactions of S100A2 and S100A6 with the Tetratricopeptide Repeat Proteins, Hsp90/Hsp70-organizing Protein and Kinesin Light Chain

Shimamoto

Takata

Tokuda

et al. 2008

S100A2 and S100A6 interact with several target proteins in a Ca 2؉-regulated manner. However, the exact intracellular roles of the S100 proteins are unclear. In this study we identified Hsp70/ Hsp90-organizing protein (Hop) and kinesin light chain (KLC) as novel targets of S100A2 and S100A6. Hop directly associates with Hsp70 and Hsp90 through the tetratricopeptide (TPR) domains and regulates Hop-Hsp70 and Hop-Hsp90 complex formation. We have found that S100A2 and S100A6 bind to the TPR domain of Hop, resulting in inhibition of the Hop-Hsp70 and Hop-Hsp90 interactions in vitro. Although endogenous Hsp70 and Hsp90 interact with Hop in resting Cos-7 cells, but not with S100A6, stimulation of these cells with ionomycin caused a Hop-S100A6 interaction, resulting in the dissociation of Hsp70 and Hsp90 from Hop. Similarly, glutathione S-transferase pulldown and co-immunoprecipitation experiments revealed that S100A6 binds to the TPR domain of KLC, resulting in inhibition of the KLC-c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1) interaction in vitro. The transiently expressed JIP-1 interacts with KLC in resting Cos-7 cells but not with S100A6. Stimulation of these cells with ionomycin also caused a KLC-S100A6 interaction, resulting in dissociation of JIP-1 from KLC. These results strongly suggest that the S100 proteins modulate Hsp70-Hop-Hsp90 multichaperone complex formation and KLC-cargo interaction via Ca 2؉ -dependent S100 protein-TPR protein complex formation in vivo as well as in vitro. Moreover, we have shown that S100A2 and S100A6 interact with another TPR protein Tom70 and regulate the Tom70-ligand interaction in vitro. Thus, our findings suggest a new intracellular Ca 2؉