Human Sgt1 Binds HSP90 through the CHORD-Sgt1 Domain and Not the Tetratricopeptide Repeat Domain

Abstract

Sgt1 has been identified as a subunit of both core kinetochore and SCF (Skp1-Cul1-F-box) ubiquitin ligase complexes and is also implicated in plant disease resistance. Sgt1 has two putative HSP90 binding domains, a tetratricopeptide repeat and a p23-like CHORD and Sgt1 (CS) domain. Using NMR spectroscopy, we show that only the CS domain of human Sgt1 physically interacts with HSP90. The tetratricopeptide repeat domain does not bind to either HSP90 or HSP70. Determination of the three-dimensional structure showed that the Sgt1-CS domain shares the same ␤-sandwich fold as p23 but lacks the last highly conserved ␤-strand in p23. Analysis of the structures of Sgt1-CS and p23 revealed a similar charge distribution on one of two opposing surfaces that suggests that it is the binding region for HSP90 in Sgt1. Although ATP is absolutely required for p23 binding to HSP90, Sgt1 binds to HSP90 also in the absence of the non-hydrolyzable analog ATP␥S. Our findings suggest the CS domain is a binding module for HSP90 distinct from p23-like domains, which implies that Sgt1 and related proteins function in recruiting heat shock protein activities to multiprotein assemblies. Heat shock protein 90 (HSP90)1 is a molecular chaperone important for protein folding. HSP90 is different from other chaperones because most of its substrates are related to signal transduction (1). Recent studies also suggest that HSP90 plays a role in protein quality control where it facilitates the polyubiquitination and degradation of substrates through interaction with the co-chaperone C terminus of HSC70-interacting protein (CHIP) (2). Thus, HSP90 can be involved in protein regulation in quite different ways depending on the cellular context.Very recently, it has been reported that Sgt1 interacts with HSP90 (3-5). Sgt1 was originally identified as a suppressor of the G 2 allele of Skp1 and was found to be important for both the G 1 /S and G 2 /M transitions in the cell cycle (6). The G 2 /M transition involves activation of the kinetochore. Sgt1 was shown to be required for the activation of the kinetochore core complex CBF3 and to physically interact with Skp1, one component of CBF3 (6). Sgt1 also physically interacts with Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes through interaction with Skp1. Moreover, a yeast Sgt1 mutant was defective in Sic1 degradation through ubiquitination (6).Sequence analysis of Sgt1 proteins from yeast, human, barley, rice, and Arabidopsis shows three conserved domains (tetratricopeptide repeat (TPR), CHORD-containing proteins and Sgt1 (CS), and Sgt1-specific (SGS)) and two variable regions (VR1 and VR2) (7). TPR domains are known as heat shock protein binding domains. The SGS domain was shown to interact with S100 calcium-binding proteins (8). The CS domain has high sequence homology with p23 and is also known as a p23-like domain (9). p23 has been shown to physically and functionally interact with HSP90, apparently serving in a role as co-chaperone. Recently, HSP90 was shown to be an essential factor required for...

Structural and Functional Analysis of SGT1 Reveals That Its Interaction with HSP90 Is Required for the Accumulation of Rx, an R Protein Involved in Plant Immunity

“…Although this and other studies have shown that the CS domain is structurally similar to cochaperone P23 (Lee et al, 2004) ( Figures 8A and 8B), our data suggest that they have distinct modes of binding to HSP90. The recent crystal structure of fulllength HSP90 in a closed conformation bound to Sba1p, the yeast homolog of P23, revealed three interaction sites ( Figure 8D): the middle domain (shown in green) and the N-terminal domain of an HSP90 monomer (light green) as well as the N-terminal domain of the other HSP90 monomer (light pink) (Ali et al, 2006).…”

“…From a series of triple resonance three-dimensional experiments, resonances arising from the backbone N, C, and H atoms were assigned together with those of the C b and H b nuclei. The secondary structures of CSa were inferred from the nuclei N, C a , C b , and H a and chemical shifts using the TALOS program (Cornilescu et al, 1999), which yielded secondary structure maps very similar to those of the CS domain from human SGT1 (Lee et al, 2004). Since both Arabidopsis and human SGT1 sequences share ;38% identity in the region spanning the CS domain, we built a three-dimensional structure of the CSa domain using homology modeling ( Figures 3A and 3B).…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…This binding interface is also used by another co-chaperone, Aha1, which binds preferentially in an asymmetric way to the closed ATP-dimer, as revealed by chemical shift perturbations and biophysical analysis, leading to the closed form of Hsp90 [195,196]. The interaction surfaces were mapped by chemical shift perturbations for further co-chaperones that bind to the N-terminal domain [197,198] and the C-terminal domain [199]. Recently, Tah1 was identified as a new Hsp90 co-chaperone in proteomic screens, linking Hsp90 to RNA Polymerase II assembly and apoptosis within mammalian cells [171,200,201].…”

The Hsp90 chaperone machinery: from structure to drug development

“…Even in the presence of the TPR domain, Sgt1 interacts with Hsp90 through the CS domain instead of the TPR domain (28)(29)(30). The recent crystal structure determination of the plant Hsp90-Sgt1 interaction revealed the binding of the Sgt1 CS domain to the N-terminal domain of Hsp90, including the most N-terminal strand region involved in stand swapping during N-terminal dimerization (30) (Fig.…”