Patterns of structural dynamics in RACK1 protein retained throughout evolution: A hydrogen‐deuterium exchange study of three orthologs

Abstract

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.

eIF6 anti-association activity is required for ribosome biogenesis, translational control and tumor progression

“…Rack1 (receptor for activated C kinase 1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins [1]. Rack1 adopts a seven-bladed β-propeller structure, which facilitates protein binding [69]. A cryo-EM study of the 80S ribosome locates Rack1 on the head region of the 40S subunit, in the immediate vicinity of the mRNA exit channel [62].…”

Capturing the Asc1p/Receptor for Activated C Kinase 1 (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast

“…As platforms or adaptors for protein-protein interactions, scaffold proteins dynamically organize protein proximities and thereby exert a major impact on cellular signaling (1,2). The G␤-like Asc1 protein from the unicellular budding yeast S. cerevisiae belongs to the WD40 family of scaffold proteins and folds into a seven-bladed ␤-propeller with a large surface for interactions (3)(4)(5). Asc1p is highly conserved throughout the eukaryotic kingdom and shares a high degree of structural similarity with its orthologs, e.g.…”

New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

“…The most pronounced decrease in backbone H/D exchange was observed for peptides 3 (16-26) and 4 (23-28), both containing Arg24, and peptides 7 (37-47) and 8 (40)(41)(42)(43)(44)(45)(46)(47), both containing Arg43 from a neighboring monomer subunit (Figure 2a, c). Other regions located close to the active site [peptides 10 (50-72), 13 (95-103), and 19 (178-182)], and peptides 1 (2-10) and 10 (50-72) from adjacent subunit are under the influence of the P i .…”

“…The resulting 60 μL of cold protein solution were further analyzed on a Waters H/DX technology platform [33] as described in Tarnowski et al [47]. A reproducible peptide list was assembled by using an in-house produced program for HD/X data analysis [48] and the Waters ProteinLynx Global Server (Waters, Milford, MA, USA).…”

“…A reproducible peptide list was assembled by using an in-house produced program for HD/X data analysis [48] and the Waters ProteinLynx Global Server (Waters, Milford, MA, USA). Hydrogen/deuterium exchange data were analyzed by the Waters DynamX 2.0 software package, permitting accurate assignment of the deuterium incorporation as described elsewhere [47].…”