RACK1 is a member of the WD repeat family of proteins and is involved in multiple fundamental cellular processes. An intriguing feature of RACK1 is its ability to interact with at least 80 different protein partners. Thus, the structural features enabling such interactomic flexibility are of great interest. Several previous studies of the crystal structures of RACK1 orthologs described its detailed architecture and confirmed predictions that RACK1 adopts a seven-bladed b-propeller fold. However, this did not explain its ability to bind to multiple partners. We performed hydrogendeuterium (H-D) exchange mass spectrometry on three orthologs of RACK1 (human, yeast, and plant) to obtain insights into the dynamic properties of RACK1 in solution. All three variants retained similar patterns of deuterium uptake, with some pronounced differences that can be attributed to RACK1's divergent biological functions. In all cases, the most rigid structural elements were confined to B-C turns and, to some extent, strands B and C, while the remaining regions retained much flexibility. We also compared the average rate constants for H-D exchange in different regions of RACK1 and found that amide protons in some regions exchanged at least 1000-fold faster than in others. We conclude that its evolutionarily retained structural architecture might have allowed RACK1 to accommodate multiple molecular partners. This was exemplified by our additional analysis of yeast RACK1 dimer, which showed stabilization, as well as destabilization, of several interface regions upon dimer formation.Keywords: receptor for activated C kinase; WD repeats; scaffolding protein; cell signaling; hydrogen deuterium exchange; mass spectrometry; protein dynamics Abbreviations: Asc1, absence of growth suppressor of Cyp1; HDXMS, hydrogen-deuterium exchange monitored by mass spectrometry; RACK1, receptor for activated C kinase 1; SEC, size exclusion chromatography; WD repeat, tryptophan aspartate repeat.

Histone pre-mRNAs are cleaved at the 3′ end by a complex that contains U7 snRNP, the FLICE-Associated Huge protein (FLASH) and Histone pre-mRNA Cleavage Complex (HCC) consisting of several polyadenylation factors. Within the complex, the N-terminus of FLASH interacts with the N-terminus of the U7 snRNP protein Lsm11 and together they recruit the HCC. FLASH through its distant C-terminus independently interacts with the C-terminal SANT/Myb-like domain of Nuclear Protein, Ataxia-Telangiectasia locus (NPAT), a transcriptional co-activator required for expression of histone genes in S-phase. To gain structural information on these interactions, we used mass spectrometry to monitor hydrogen/deuterium (H/D) exchange in various regions of FLASH, Lsm11 and NPAT alone or in the presence of their respective binding partners. Our results indicate that the FLASH-interacting domain in Lsm11 is highly dynamic, while the more downstream region required for recruiting the HCC exchanges deuterium slowly and likely folds into a stable structure. In FLASH, a stable structure is adopted by the domain that interacts with Lsm11 and this domain is further stabilized by binding Lsm11. Notably, both H/D exchange experiments and in vitro binding assays demonstrate that Lsm11, in addition to interacting with the N-terminal region of FLASH, also contacts the C-terminal SANT/Myb-like domain of FLASH, the same region that binds NPAT. However, while NPAT stabilizes this domain, Lsm11 causes its partial relaxation. These competing reactions may play a role in regulating histone gene expression in vivo.

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