In addition, the average centrality of the Rar1-CHORD2 residues is higher than in the other domains, i.e. the key mediator residues are surrounded by “supporting cast” residues that have sufficient communication capacity to rapidly disseminate the information signal from central mediators across the network. Hence, the Rar1-CHORD2 binding could regulate the long-range interactions and promote stabilization of the ternary complex. The network analysis could thus clarify the elusive role of Rar1 as a key component of the ternary assembly and stability enhancer of the Hsp90-cochaperone interactions. Overall, our results support the mechanism in which a selected group of critical Rar1CHORD2 residues, particularly Rar1-W217 as a potential principal contributor, may be critical for mediating long-range interactions and modulation of the Hsp90-ATPase activity. Our results indicated that the Rar1-CHORD2 binding may result in a more assortative interaction network that is better integrated through preferential association of global mediating residues with many locally connected hubs. The centrality analysis of the Hsp90-Cdc37 Praeruptorin-B complex similarly revealed that key mediating residues and functional hot spots are aligned with the peaks in the closeness and betweenness profiles. The characteristic peaks were conserved in both distributions and corresponded in the Hsp90NTD to the residues E47 and Q133. Both residues form stabilizing polar interactions with R167 in Cdc37 and these interactions are implicated as a major contributing factor in the mechanism of Cdc37-mediated inhibition of the ATPase activity. The centrality analysis recovered major functional sites E47, Q133, and F134 as key mediating residues of allosteric communications in the Hsp90-Cdc37 complex. Interestingly, E47A mutation reduced Hsp90-Cdc37 binding by 50%, while Q133A mutation could decrease the Hsp90-Cdc37 interactions by 85%. While our analysis 14alpha-hydroxy-Sprengerinin-C robustly selected the Hsp90 binding hot spots, the simplicity of the centrality metric somewhat underestimated the relative contribution of Q133 to the Hsp90-Cdc37 binding that is more significant that was predicted. In the Cdc37MD, the centrality peaks corresponded to R167 and Q208 residues that were also among highly connected interfacial hubs. Overall, the centrality analysis suggested that the group of strongly interacting residues Hsp90-E47, Hsp90-Q133, Cdc37-R167, and Cdc37-Q208 could collectively form a major gateway for allosteric communications in the Hsp90-Cdc37 complex. These global mediating residues are also highly connected local hubs and major contributors of the structurally stable communities. The prominent role of these residues in the Hsp90-Cdc37 binding was demonstrated via a comprehensive biochemical analysis in which mutations of Hsp90-Q133, Cdc37-R167 and Cdc37-Q208 essentially abolished the complex formation, causing 85%�C90% reduction in the Hsp90-Cdc37 interactions as measured in cell-based assays. These key functional sites were consistently recovered as largest peaks in both closeness and betweenness metrics. Mutations of key mediating nodes in the Hsp90-Cdc37 complex would cause a simultaneous disruption of multiple interactions and disrupt the integrity of the allosteric network, thus leading to a dramatic loss of the chaperone activity. At the same time, the centrality analysis unveiled a few smaller noticeable peaks corresponding to the Hsp90-NTD residues Y61, L91 W162 and Cdc37-F238. These residues are not located at the interdomain interface and reside within their respective domains, contributing to the structural integrity of the interacting modules.