Structural Determinants of Natriuretic Peptide Receptor Specificity and Degeneracy

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Abstract

Cardiovascular homeostasis and blood pressure regulation are reliant, in part, on interactions between natriuretic peptide (NP) hormones and natriuretic peptide receptors (NPR). The C-type NPR (NPR-C) is responsible for clearance of NP hormones from the circulation, and displays a cross-reactivity for all NP hormones (ANP, BNP, and CNP), in contrast to other NPRs, which are more restricted in their specificity. In order to elucidate the structural determinants for the binding specificity and cross-reactivity of NPR-C with NP hormones, we have determined the crystal structures of the complexes of NPR-C with atrial natriuretic peptide (ANP), and with brain natriuretic peptide (BNP). A structural comparison of these complexes, with the previous structure of the NPR-C/CNP complex, reveals that NPR-C uses a conformationally inflexible surface to bind three different, highly flexible, NP ligands. The complex structures support a mechanism of rigid promiscuity rather than conformational plasticity by the receptor. While ANP and BNP appear to adopt similar receptor-bound conformations, the CNP structure diverges, yet shares sets of common receptor contacts with the other ligands. The degenerate versus selective hormone recognition properties of different NPRs appears to derive largely from two cavities on the receptor surfaces, pocket I and pocket II, that serve as anchoring sites for hormone side-chains and modulate receptor selectivity.

Introduction

Natriuretic peptide (NP)/natriuretic peptide receptor (NPR) interactions play key roles in cardiovascular homeostasis and blood pressure regulation.1 The three members of the NP family are atrial (ANP), brain (BNP) and C-type (CNP, mainly expressed in endothelial cells) natriuretic peptides.2 These peptides counterbalance the renin-angiotensin system by increasing natriuresis and diuresis.3 The action of NPs is mediated through their cell-surface receptors, NPRs, which are a family of at least three homologous single-transmembrane, glycosylated receptors (NPR-A, NPR-B, NPR-C).2,4., 5., 6. NPR-A and NPR-B, also called guanylyl cyclase A (GC-A) and guanylyl cyclase B (GC-B), transduce the NP signal through a large cytoplasmic domain with guanylyl cyclase activity.5 The NPR-C receptor, which primarily serves as a “clearance” receptor5 to remove NP hormones from the circulation, contains a 37 amino acid residue cytoplasmic tail, which reportedly is phosphorylated and engages Gi-proteins in a hormone-dependent fashion, however the precise details of NPR-C signaling remain unclear.7., 8., 9., 10., 11. Despite their different downstream signaling cascades, the NPR family receptors share a similar extracellular domain (ECD) of about 450 amino acid residues with ∼30% homology and conserved topology, which is responsible for recognizing and binding NPs.4 Different NPRs can bind the same ligand, e.g. both NPR-A and NPR-C bind ANP and BNP, both NPR-B and NPR-C bind CNP, indicating that the extracellular segments of all NPRs have similar ligand recognition and activation mechanisms.

The NP receptors pose some interesting specificity and cross-reactivity questions (Table 1). NPR-C is the most promiscuous of the three receptors, binding to all NPs with high affinity,12, but NRP-A and NPR-B are more specific towards their own spectrums of ligands.4,13,14 The rank order of affinities between NPRs and NPs are ANP > BNP  CNP for NPR-A, CNP  ANP > BNP for NPR-B, and ANP > CNP > BNP for NPR-C respectively (see Table 1). NPR-A is the most favored binding partner for ANP, and NPR-B is almost exclusively specific for CNP. Extensive structure-activity studies of NPs have converged on the idea that the residues within the cyclized loop are largely, although not completely, responsible for receptor selectivity, whereas the flanking residues outside the ring can modulate affinity.12,15., [16], 17. The mechanism by which the NPR-C surface engages different NP residues is not well understood structurally.

To-date, four crystal structures have been solved of NPR. These structures are the unliganded NPR-A extracellular domain (ECD),18 the unliganded NPR-C ECD and the NPR-C ECD complexed with CNP,19 and lately, the NPR-A ECD/ANP complex.20 The overall structures of NPR-A and NPR-C extracellular domains both display the same dumbbell-shaped periplasmic binding protein scaffold, with a bound chloride ion located in each membrane-distal domain. Structure determination of the apo NPR-C and ligand-complexed NPR-C structures clarified that the ECDs of NPRs dimerize through their membrane-distal domains, and one NP hormone binds in between two NPR receptor monomers with 1:2 hormone:receptor stoichiometry.19 From comparison of bound versus free receptor structures of NPR-C, it was obvious that a large hormone-induced conformational change, that brings the membrane-proximal domains of the dimer closer by about 20 angstroms, occurred upon ligand binding. The recently determined structure of the NPR-A/ANP complex also shows the 1:2 hormone:receptor stoichiometry and a large-scale ANP-induced conformational change. However, it differs in that there is more of an inter-dimer rotational twist, rather than the straight hinge-like dimer closure seen in NPR-C.20 The different types of conformational change may reflect the different signaling properties of NPR-A versus NPR-C, whose signaling ability remains to be completely defined. Collectively, the NPR/NP complexes revealed the general scheme of NP/NPR interaction, but little has been known about the structural basis of binding selectivity between NP and NPRs. Given the important clinical role that BNP has assumed for treatment of congestive heart failure, studies are needed to address the structural features underlying capability of NPR-C as degenerate NP clearance receptor versus the narrow ligand-binding spectrums of other NPRs. Such information will assist therapeutic strategies to extend the half-life of BNP in the bloodstream through selective antagonism of NPR-C clearance.

Here, we have determined the crystal structures of the NPR-C/ANP complex and the NPR-C/BNP complex. By examining the structural details of NPR-C binding to three different NP ligands and comparing the available structural data, we have gained insights into the NPR/NP interaction and the structural determinants of ligand/receptor binding specificity in this important receptor family.

Section snippets

Structural determination

The human NPR-C extracellular domain was expressed and purified from Drosophila S2 cells. The NPR-C/NP complexes were prepared by mixing NPR-C with excess human ANP or human BNP, and re-purified by size-exclusion chromatography. The crystals are isomorphous with those of NPR-C/CNP, albeit with slightly varied unit cell dimensions (Table 2). The asymmetric unit of these crystals contains a full NPR-C/NP complex, i.e. two copies of NPR-C and one copy of NP, with a non-crystallographic 2-fold axis

Cloning and expression

The peptide hormones ANP, BNP, and CNP were purchased from BACHEM (South San Francisco, CA). These peptides were dissolved in water to 5 mg/ml before being mixed with receptor proteins. The procedures for cloning and expressing the extracellular domains of human NPR-C from D. melanogaster S2 expression system were as described.19 Briefly, the coding sequence for human NPR-C plus a six-histidine affinity-tag was cloned into the pRMHa3 vector; the construct was co-transfected in S2 cells with a

Acknowledgements

We thank Dar-chone Chow, Focco van den Akker, and Monika Martick for assistance in cell culture and calorimetry, and the Stanford Synchrotron Radiation Laboratory for support in X-ray data collection. K.C.G. is supported by the NIH, Keck and HHMI. X.L.H. is supported by the Fritz Krauth Memorial Postdoctoral Fellowship from Paralyzed Veterans of America/Spinal Cord Research Foundation.

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    Present address: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, 303 E Chicago Ave, Searle 8-416, Chicago, IL 60611, USA.

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