Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry

Abstract

Defining protein-protein contacts is a challenging problem and cross-linking is a promising solution. Here, we present a case of mitochondrial single strand binding protein Rim1 and helicase Pif1, an interaction first observed in immuno-affinity pull-down from yeast cells using Pif1 bait. We found that only the short succinimidyl-diazirine cross-linker or formaldehyde captured the interaction between recombinant Rim1 and Pif1. In addition, Pif1 needed to be stripped of its N-terminal and C-terminal domains, and Rim1's C-terminus needed to be modified for the cross-linked product to become visible. Our report is an example of a non-trivial analysis, where a previously identified stable interaction escapes initial capture with cross-linking agents and requires substantial modification to recombinant proteins and fine-tuning of the mass spectrometry-based methods for the cross-links to become detectable. We used high resolution mass spectrometry to detect the cross-linked peptides. A 1:1 mixture of 15N and 14N-labeled Rim1 was used to validate the cross-links by their mass shift in the LC-MS profiles. Two sites on Rim1 were confirmed: 1) the N-terminus, and 2) the K29 residue. Performing cross-linking with a K29A variant visibly reduced the cross-linked product. Further, K29A-Rim1 showed a five-fold lower affinity to single stranded DNA compared to wild-type Rim1. Both the K29A variant and wild type Rim1 showed similar degrees of stimulation of Pif1 helicase activity. We propose structural models of the Pif1-Rim1 interaction and discuss its functional significance. Our work represents a non-trivial protein-protein interface analysis and demonstrates utility of short and non-specific cross-linkers.

The mitochondrial single-stranded DNA binding protein from S. cerevisiae, Rim1, does not form stable homo-tetramers and binds DNA as a dimer of dimers

“…We note that Zybailov et al. ( 40 ) reported having solved the structure for Rim1. However, no information has been made available yet and it remains to be determined whether the two crystal forms we detected are the only two forms that Rim1 can adopt.…”

“…Therefore, it remains to be determined whether interaction of Pif1 occurs with a tetramer or dimer of Rim1. Of note, the Rim1–Pif1 interaction was shown to be lost upon Rim1 binding to a 70 nt ssDNA ( 40 ), making it difficult to rationalize the stimulatory effect of Rim1 on the unwinding activity of Pif1 ( 8 ). If Pif1 were to bind to a dimer of Rim1, loss of Rim1–Pif1 interaction upon binding of DNA to Rim1 may be explained by the highly cooperative tetramerization of Rim1 on such a long ssDNA (Figure 6 , complex D).…”

Interaction of Isocitrate Lyase with Proteins Involved in the Energetic Metabolism in Paracoccidioides lutzii

“…The KFC-2 server provided means to identify the most common residues involved in the interaction of ICL with other proteins. Finding hot spots on the protein surface has shed light on experimental applications in the biological fields [ 79 , 80 , 81 ] and understanding PPIs means understanding proteins’ biological functions. Here, we used this approach in order to identify the main contact preference regions of P. lutzii ICL when it interacted with proteins from mycelium, mycelium-to-yeast and yeast phases.…”

Role and Regulation of Pif1 Family Helicases at the Replication Fork

“…Full-length ScPif1 preferentially unwinds RNA-DNA hybrid duplexes with the RNA in the displaced strand [ 34 ] due to increased processivity on the RNA-DNA duplex [ 35 ], but this enhanced activity on RNA-DNA duplexes is lost when the NTD is removed [ 29 ]. The NTD of ScPif1 is required in order for the mitochondrial single-stranded binding protein, Rim1, to enhance the unwinding activity of ScPif1 [ 36 ], even though direct interactions between the helicase domain of ScPif1 and Rim1 have been reported [ 37 ]. The strand annealing activity of hPIF1 resides in the NTD [ 30 ].…”