title: 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 of15N and14N-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Boris
authors.initials: B
authors.lastname: Zybailov
authors.affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR-72205, USA.
authors.forename: Kuppan
authors.initials: K
authors.lastname: Gokulan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Jadon
authors.initials: J
authors.lastname: Wiese
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Ramanagouda
authors.initials: R
authors.lastname: Ramanagoudr-Bhojappa
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Alicia K
authors.initials: AK
authors.lastname: Byrd
authors.affiliation: Department of Biomedical Informatics, University of Arkansas for Medical Sciences, USA.
authors.forename: Galina
authors.initials: G
authors.lastname: Glazko
authors.affiliation: UALR/UAMS joint bioinformatics program, University of Arkansas Little Rock, Little Rock, AR, USA.
authors.forename: Mihir
authors.initials: M
authors.lastname: Jaiswal
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Mackintosh
authors.affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR-72205, USA.
authors.forename: Kottayil I
authors.initials: KI
authors.lastname: Varughese
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Raney
keywords: DNA-helicase; Mass spectrometry (MS); Protein cross-linking; Protein-DNA interaction; Protein-protein interaction