Investigating the human Calcineurin Interaction Network using the πɸLxVP SLiM

Abstract

Ser/thr phosphorylation is the primary reversible covalent modification of proteins in eukaryotes. As a consequence, it is the reciprocal actions of kinases and phosphatases that act as key molecular switches to fine tune cellular events. It has been well documented that ~400 human ser/thr kinases engage substrates via consensus phosphosite sequences. Strikingly, we know comparatively little about the mechanism by which ~40 human protein ser/thr phosphatases (PSPs) dephosphorylate ~15000 different substrates with high specificity. The identification of substrates of the essential PSP calcineurin (CN) has been exceptionally challenging and only a small fraction has been biochemically confirmed. It is now emerging that CN binds regulators and substrates via two short linear motifs (SLiMs), the well-studied PxIxIT SLiM and the LxVP SLiM, which remains controversial at the molecular level. Here we describe the crystal structure of CN in complex with its substrate NFATc1 and show that the LxVP SLiM is correctly defined as πɸLxVP. Bioinformatics studies using the πɸLxVP SLiM resulted in the identification of 567 potential CN substrates; a small subset was experimentally confirmed. This combined structural-bioinformatics approach provides a powerful method for dissecting the CN interaction network and for elucidating the role of CN in human health and disease.

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

“…Use of such regular expressions (RegEx) identifies only exact sequence matches and treats information at all motif positions equally. Limitations of this approach are illustrated by a previous effort to identify LxVP-containing proteins using a structure-based RegEx that fails to identify 16 of 25 LxVP-containing peptides discovered here via ProP-PD (Sheftic et al, 2016). Similarly, RegEx's used for PxIxIT identification exclude information from flanking residues, which significantly influence binding affinity (Nguyen et al,bioRxiv 306779) and are represented in the 12 position PSSM developed here.…”

“…Mutation of PxIxIT or LxVP sequences in substrates or their conserved binding surfaces on CN disrupts dephosphorylation, and many CN inhibitors, including CsA, FK506, and the viral A238L protein, function by blocking SLiM binding (Grigoriu et al, 2013). Although these SLiMs have been characterized at the structural level (Li et al, 2004;Sheftic et al, 2016), the small number of validated PxIxIT and LxVP instances in humans has limited our understanding of the specificity determinants of the CN SLiM-binding pockets and hampered global identification of these motifs.…”

Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20

“…Active APC/C Cdc20 liberated from its inhibitor XErp1 subsequently targets cyclin B and securin for degradation resulting in sister chromatid segregation and meiotic exit. So far, we could not identify known binding motifs of CaN on XErp1 and Cdc20, e.g., “PxIxIT” or “LxVP” motifs . Thus, future studies will be important to decipher the exact binding mode of CaN to Cdc20 and XErp1.…”

Molecular basis of calmodulin-dependent calcineurin activation

“…Our current understanding of the protein's activation and enzymatic activity has been shaped by a number of atomic resolution structures of CaN determined by X-ray crystallography [4][5][6][7][8][9] and nuclear magnetic resonance spectroscopy [10]. Of the many CaN structures that have been deposited to the Protein Data Bank are structures that have revealed the protein's auto-inhibited state (PDB ID: 1aui [4]), a potentially non-physiological 2:2 CaM/CaN stoichiometric configuration [7,11,12], and complexes of the enzyme with immunosuppressants [5,8] and transcription factors [6,9]. However, much less is known about the structural basis of CaM-dependent regulation of CaN, as atomic resolution CaM/CaN complexes are limited to the intact CaM bound to small peptides comprising the CaM binding region (CaMBR) of the CaN regulatory domain [13].…”