Structural probing of Hsp26 activation and client binding by quantitative cross-linking mass spectrometry
Small heat-shock proteins (sHSP) are important members of the cellular stress response in all species. Their best described function is the binding of early unfolding states and the resulting prevention of protein aggregation. Most sHSPs exist as oligomers but vary in size and subunit organization. Many sHSPs exist as a polydisperse composition of oligomers which undergoes changes in subunit composition, folding status and relative distribution upon heat activation. To date only an incomplete picture of the mechanism of sHSP activation exists and in particularly the molecular basis of how sHSPs bind client proteins and mediate client specificity is not fully understood. In this study we have applied cross-linking mass spectrometry (XL-MS) to obtain detailed structural information on sHSP activation and client binding for yeast Hsp26. Our cross-linking data reveals the middle domain of Hsp26 as client-independent interface in multiple Hsp26::client complexes and indicates that client-specificity is likely mediated via additional binding sites within its CD and CTE. Our quantitative XL-MS data underpins the middle domain as the main driver of heat-induced activation and client binding but shows that global rearrangements spanning all domains of Hsp26 are taking place simultaneously. We also investigated a Hsp26::client complex in the presence of Ssa1 (Hsp70) and Ydj1(Hsp40) at the initial stage of refolding and observe that the interaction between refolding chaperones is altered by the presence of a client protein, pointing to a mechanism where interaction of Ydj1 with the HSP::client complex initiates assembly of the active refolding machinery.