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My accidental encounter with protein hydrogen exchange (HX) at its very beginning and its continued development through my scientific career have led us to a series of advances in HX measurement, interpretation, and cutting edge biophysical applications. After some thoughts about how life brought me there, I take the opportunity to reflect on our early studies of allosteric structure and energy change in hemoglobin, the still-current protein folding problem, and our most recent forward-looking studies on protein machines. 

WNTs play key roles in development and disease, by binding both Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors that include the ROR and RYK receptor tyrosine kinases (RTKs). We describe crystal structures and WNTbinding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2). RORs bind WNTs though a FZD-related cysteine-rich domain (CRD), and RYKs through a WNT-inhibitory factor (WIF) domain. Our structures suggest that neither the Nrk CRD nor the Drl-2 WIF domain can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable with a WNT acyl chain. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also show that DWnt-5, which regulates Drosophila ROR and RYK orthologs, lacks an acyl chain. Together with analysis of WNT/receptor interaction sites, these structures provide new insight into how WNTs recruit their RTK co-receptors into signaling complexes. 

Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution. 

Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

 

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.