In silico protein structural analysis of PRMT5 and RUVBL1 mutations arising in human cancers

Abstract

DNA double strand breaks (DSBs) can be generated spontaneously during DNA replication and are repaired primarily by Homologous Recombination (HR). However, efficient repair requires chromatin remodeling to allow the recombination machinery access to the break. TIP60 is a complex conserved from yeast to humans that is required for histone acetylation and modulation of HR activity at DSBs. Two enzymatic activities within the TIP60 complex, KAT5 (a histone acetyltransferase) and RUVBL1 (an AAA+ ATPase) are required for efficient HR repair. Post-translational modification of RUVBL1 by the PRMT5 methyltransferase activates the complex acetyltransferase activity and facilitates error free HR repair. In S. pombe a direct interaction between PRMT5 and the acetyltransferase subunit of the TIP60 complex (KAT5) was also identified. The TIP60 complex has been partially solved experimentally in both humans and S. cerevisiae, but not S. pombe. Here, we used in silico protein structure analysis to investigate structural conservation between S. pombe and human PRMT5 and RUVBL1. We found that there is more similarity in structure conservation between S. pombe and human proteins than between S. cerevisiae and human. Next, we queried the COSMIC database to analyze how mutations occurring in human cancers affect the structure and function of these proteins. Artificial intelligence algorithms that predict how likely mutations are to promote cellular transformation and immortalization show that RUVBL1 mutations should have a more drastic effect than PRMT5. Indeed, in silico protein structural analysis shows that PRMT5 mutations are less likely to destabilize enzyme function. Conversely, most RUVBL1 mutations occur in a region required for interaction with its partner (RUVBL2). These data suggests that cancer mutations could destabilize the TIP60 complex. Sequence conservation analysis between S. pombe and humans shows that the residues identified in cancer cells are highly conserved, suggesting that this may be an essential process in eukaryotic DSB repair. These results shed light on mechanisms of DSB repair and also highlight how S. pombe remains a great model system for analyzing DSB repair processes that are tractable in human cells.

Keywords: Cancer genetics; Chromatin remodeling; DNA damage; Mutation; Protein structural analysis.

Fig. 1.. Alignments of human and yeast protein AlphaFold structures.

AlphaFold structure models are shown for A) human PRMT5 in gray, B) S. pombe Skb1 in magenta, C) S. cerevisiae HSL7, D) human RUVBL1 in dark gray and RUVBL2 in light gray, E) S. pombe Rvb1 in magenta and Rvb2 in light magenta, F) S. cerevisiae RUVBL1 in cyan and RUVBL2 in light cyan. G) S. pombe Skb1 in magenta and S. cerevisiae HSL7 in cyan are aligned to human PRMT5 in gray. H) S. pombe Rvb1-Rvb2 in magenta (dark magenta for Rvb1 and light pink for Rvb2) and S. cerevisiae RUVBL1-RUVBL2 in cyan (dark cyan for RUVBL1 and light cyan for RUVBL2) are aligned to human RUVBL1-RUVBL2 in gray (dark gray for RUVBL1 and light gray for RUVBL2). Alignments and resulting RMSDs were calculated using PyMOL.

Fig. 2.. RUVBL1 COSMIC mutations.

A. Distribution of all reported RUVBL1 mutations on COSMIC. B. RUVBL1 coding mutation density. A histogram of mutations was overlayed on a diagram of RUVBL1 adapted from [45,59]. C. Incidence of PRMT5 and RUVBL1 mutations in human cancers. D. RUVBL1 and PRMT5 mutations. The figure shows the position of RUVBL1 truncating mutations (black) and high frequency RUVBL1 and PRMT5 mutations (red). PRMT5 truncating mutations were reported previously [66]. Diagramed on the figure are also three pairs of co-occurring mutations (green, blue, yellow) that are predicted to affect the structure of RUVBL1 (please see text).

Fig. 3.. The K274 residue found in the RUVBL1 structure interacts with both RUVBL2 and EP400.

A) An overview of the human RUVBL1-RUVBL2 hexamer interacting with EP400 from PDB 8XVG. The RUVBL1 subunits are shown in silver, the RUVBL2 subunits in gold, and the EP400 protein is shown in dark pink. B) The K274 residue of RUVBL1 (silver) is shown in cyan sticks forming a hydrogen bond (green dashed line) with L260 of RUVBL2 (gold). C) The K274 residue of RUVBL1 (silver) is shown in cyan sticks forming a hydrogen bond (green dashed line) with Q1748 of EP400 (dark pink).