ABSTRACT
Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.
Subject(s)
Adenosine Triphosphatases/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Protein Domains/genetics , Adenosine Triphosphatases/genetics , Glutamic Acid/genetics , HEK293 Cells , HSP90 Heat-Shock Proteins/genetics , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation/physiology , Structure-Activity Relationship , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
The activity of the tumor suppressor p53 has to be timed and balanced closely to prevent untimely induction of cell death. The stability of p53 depends on the ubiquitin ligase Mdm2 but also on Hsp70 and Hsp90 chaperones that interact with its DNA binding domain (DBD). Using hydrogen exchange mass spectrometry and biochemical methods, we analyzed conformational states of wild-type p53-DBD at physiological temperatures and conformational perturbations in three frequent p53 cancer mutants. We demonstrate that the Hsp70/Hdj1 system shifts the conformational equilibrium of p53 toward a flexible, more mutant-like, DNA binding inactive state by binding to the DNA binding loop. The analyzed cancer mutants are likewise destabilized by interaction with the Hsp70/Hdj1 system. In contrast, Hsp90 protects the DBD of p53 wild-type and mutant proteins from unfolding. We propose that the Hsp70 and Hsp90 chaperone systems assume complementary functions to optimally balance conformational plasticity with conformational stability.
Subject(s)
HSP40 Heat-Shock Proteins/chemistry , Neoplasms/genetics , Protein Conformation , Tumor Suppressor Protein p53/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , HSP40 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/genetics , Humans , Mass Spectrometry , Molecular Chaperones , Neoplasms/pathology , Protein Domains/genetics , Protein Unfolding , Tumor Suppressor Protein p53/geneticsABSTRACT
Therapeutic monoclonal antibodies (mAbs) represent a milestone in pharmacological development. Their superiority is based on the combination of high specificity, low toxicity, and long half-life that characterizes biologics. If biologics have Achilles' heel, it is their potential immunogenicity. To better understand the impact of the size of immune complexes of mAbs on anti-drug antibody (ADA) dependent adverse reactions in Macaca fascicularis, we developed an efficient high-throughput size exclusion chromatography- (SEC-) based methodology that enables analysis of the size, size distribution, and ratio of free and ADA-complexed mAb in serum allowing for assessment of formation and clearance of circulating ADA-mAb immune complexes (CIC).
