In this study we have defined the contribution of specific residues of the NFAT-derived LxVP binding site to CN binding and have identified a hydrophobic pocket in CN where LxVP binds. This pocket also mediates the interaction of several yeast CN targets, including Rcn1. Our results thus suggest that partners interact with CN through two important routes, the PxIxT motif and the LxVP motif, which appears to be a conserved mode of substrate-CN interaction. Our analyses also provide insights into the mechanism by which immunosuppressants inhibit CN, showing that IS-IP complexes compete for binding to the same docking surface in CN that mediates LxVP-type interactions with natural substrates.
In yeast, PxIxIt motifs regulate the affinities of several CN substrates, thereby determining the calcium concentration dependence and in vivo signaling output of CN (Roy et al., 2007
). The availability of CN in yeast cells is limiting, so substrate selection and the profile of signaling pathways activated downstream of CN are determined by relative binding affinities (Roy et al., 2007
). Our data show that conserved CN residues essential for LxVP-binding are required for interaction of yeast CN with substrates and Rcn1, Ca2+
-activated transcription of Crz1, and the ability of yeast cells to survive stress. Thus, we propose that CN-interacting proteins contain regions with LxVP-type activity of varying affinities for CN, which, in cooperation with PxIxIT motifs, establish the overall affinity of each substrate or regulator for CN, and determine calcium dependence and signaling output.
Among yeast CN partners, only Rcn1, a member of the RCAN family of CN regulators, contains a readily identifiable LxVP motif (Görlach et al., 2000
; Hogan et al., 2003
; Kingsbury and Cunningham, 2000
). This may indicate degeneracy in the sequence requirements for binding to the BBD-CnB composite site, so that CN-binding motifs are not easily detected by sequence comparison. Data presented here, together with earlier work, identify key components of the LxVP motif in NFAT, and show that amino acids flanking the LxVP core, including Y3, are critical for CN binding (Park et al., 2000
). Since all known functional LxVP motifs have an aromatic residue at this position, we can redefine an extended core sequence, ΦLxVP, where x cannot be glycine. These insights will be critical in identifying functional ΦLxVP motifs in other CN partners, both mammalian and fungal.
It is possible that the CN-binding surface on some substrates is formed by conformational association of separate regions of primary sequence. This scenario is reminiscent of immunosuppressant-immunophilin complexes, in which three dimensional structures that specifically bind to the BBD-CnB composite site are created fortuitously by the association of distinct immunophilin residues with the structurally unrelated FK506 and CsA molecules (Ke and Huai, 2003
). This raises the possibility that the BBD-CnB composite site acts as a shared docking surface for substrates with dissimilar sequences. Further analysis is needed to identify the motifs or tertiary structures in these substrates that confer LxVP-like functions.
In mammals, NFATs mediate transcriptional activation in many CN-dependent processes (Crabtree and Olson, 2002
; Hogan et al., 2003
), and there is strong evidence that LxVP and PxIxIT motifs in NFAT proteins cooperate to determine their overall affinity for CN. The PxIxIT motif plays a major role in determining NFAT-CN interaction (Aramburu et al., 1998
), and binds to the phosphatase domain of inactive CN at a region distinct from the active site (Aramburu et al., 1998
; Li et al., 2007
). Our earlier work showed that the LxVP motif also contributes to docking, since its deletion reduces NFATc1 binding to CN (Martínez-Martínez et al., 2006
), and here we show that NFATc1 binding is inhibited by mutations in the CN hydrophobic pocket. In vitro, NFAT binding to CN increases substantially upon CN activation, an effect that is independent of phosphatase activity and is inhibited by the FK506-FKBP12 complex (Garcia-Cozar et al., 1998
). These findings suggested the presence of PxIxIT-independent contacts that might increase affinity or restrict the orientation of the substrate relative to CN (Li et al., 2007
). Indeed, it has been proposed that a docking site near the CN active site is unmasked when CN is activated (Garcia-Cozar et al., 1998
). The docking surface we identify here fulfils the requirements of this proposed site, since LxVPc1 binds near the active site and does not bind inactive CN. Our results thus support the idea that the CnA-CnB composite surface is responsible for the increased binding of NFAT to active CN. Unlike protein kinases, phosphatases do not target stringent sequence motifs (Agostinis et al., 1990
). The activity of CN on short peptides is very low, and addition of residues N-terminal to the phospho-site increases the dephosphorylation rate (Blumenthal et al., 1986
; Donella-Deana et al., 1994
), suggesting that substrate motifs which neighbor target phospho-residues can increase interaction efficiency. Substrates and regulators of protein phosphatase 1 including DARPP32 and inhibitor 1 require two distinct interaction domains to bind this phosphatase (Kwon et al., 1997
). Our studies of calcineurin suggest recognition of substrates through multiple interaction domains may be a general feature of protein phosphatases.
Since no data are available on the structure of LxVP-CN, we modeled possible structural configurations with the HADDOCK program. Molecular dynamic (MD) simulations were performed on the most likely docking structures, estimating the relative free energies of binding (details in Supplemental Data
). LxVPc1 is almost parallel to the CnB-binding α-helix of CnA, with the YLAVP core sits in the hydrophobic pocket formed by W352, P355 and F356 (). The MD simulation suggests that these residues are responsible for the electrostatic and van der Waals interactions between LxVPc1 and CN. This study predicts that the proline of LxVP interacts with aromatic residues in CN, a common feature in proline-binding domains (Zarrinpar et al., 2003
). Such proline-based interactions are weak but specific, and almost all have additional binding sites for recognizing substrates. Thus the docking surface in CN may act as a proline-recognition domain important for specific substrate recognition.
Predicted binding mode of LxVP based on docking and molecular dynamic simulations
IP-IS complexes are large structures and since they bind to a CN region close to the active site, inhibition was first proposed to involve occlusion of the active site by regions not directly involved in CN binding (Griffith et al., 1995
; Kissinger et al., 1995
). The hydrophobic pocket we identify at the interface of the CnA and CnB subunits mediates binding not only to CN substrates but also to CsA and FK506 immunophilin complexes. These complexes compete with LxVP for CN, suggesting that they bind to the same docking surface on CN. Although we cannot discard the occlusion model, our results indicate that blockade of the interaction between CN and LxVP-type structures in substrates is sufficient to explain immunosuppressant inhibition of CN.
Many of the severe side effects of immunosuppressants (such as neurotoxicity, diabetes, nephrotoxicity and hypertension) are at least partly independent of CN (Kiani et al., 2000
; Martínez-Martínez and Redondo, 2004
). These actions preclude use of these drugs for chronic inflammatory conditions and other diseases. Therefore identifying selective CN inhibitors that avoid these secondary effects is of high interest. The common CN docking pocket for substrates, regulators and IP-IS complexes presents a target for the development of immunophilin-independent immunosuppressive drugs that would avoid some side effects associated with the FK506 and CsA. Such drugs would also be valuable for exploring important CN-regulated processes such as cell-stress responses, immune activation and cardiac hypertrophy.