Structural Probing of Hsp26 Activation and Client Binding by Quantitative Cross-Linking Mass Spectrometry

Small heat-shock
proteins (sHSPs) 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. 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;
in particular, 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 a client-independent interface in multiple Hsp26::client
complexes and indicates that client specificity is likely mediated
via additional binding sites within its α-crystallin domain
and C-terminal extension. 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 take 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 the interaction of Ydj1 with the HSP::client
complex initiates the assembly of the active refolding machinery.