Conformational Analysis of Processivity Clamps Demonstrates that Protein Tertiary Structure Does Not Correlate with Dynamics

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Processivity clamps are critical for efficient DNA replication in all organisms. Whereas these clamp proteins can be either dimers or trimers, they all exhibit pseudo-six-fold symmetry. Each pseudo-monomeric domain is highly conserved in its tertiary structure even though the primary structures are divergent. We previously characterized the beta processivity clamp from Escherichia coli using hydrogen-deuterium exchange mass spectrometry (HXMS), which probes the solvent accessibility and hydrogen bonding of each backbone amide hydrogen, except those in prolines and at the first residue. We found that the three different domains within each monomer displayed different dynamics and that Domain I, which dissociates from Domain III to open the clamp, underwent partially cooperative local unfolding with a half-life of ∼4 h. To determine how general our observations of a highly dynamic clamp protein were, we carried out a similar analysis using HXMS to characterize the dynamics of clamps from bacteriophage T4, the yeast Saccharomyces cerevisiae, archaeon Thermococcus kodakarensis, plant Arabidopsis thaliana, and human. Thermococcus kodakarensis and Arabidopsis thaliana both have two different PCNA clamp proteins that show slightly different dynamics. This is especially intriguing in the case of the two PCNA proteins from Arabidopsis thaliana, as the primary structures are 96% identical. The different clamps show a wide range of dynamic properties. Bacteriophage T4 gp45 shows high deuterium uptake and undergoes widespread local unfolding events with half-lives of approximately 5 min. In human PCNA, local unfolding is observed at the trimer interfaces with a half-life of about 1 h. The clamp proteins from bacteriophage T4, Arabidopsis thaliana, and human incorporate the most deuterium and are therefore the most dynamic. We find a striking range of dynamic properties of the clamp proteins despite their highly conserved tertiary structures.