The conformation of porcine‐brain natriuretic peptide by two‐dimensional NMR spectroscopy

Abstract

Two-dimensional (2D) 'H-NMR spectra of porcine-brain natriuretic peptide (pBNP) have been recorded at 300 MHz and 400 MHz. Peak assignments have been made and the combined information from chemical shifts, coupling constants, temperature coefficients and NOES have been used to determine the conformational properties of pBNP in (C2H3)2S0. Overall the peptide appears to be flexible, with the possibility of some P-type structure near the C terminus. Some of the assignments and deduced structural features in the current study differ from those in a recent report by Inooka et al. [Inooka, H., Kikuchi, T., Endo, S., Ishibashi, Y., Wakimasu, M. and Mizuta, E. (1990) Eur. J. Biochem. 193,127-1341 which may indicate the sensitivity of the structure of this peptide to differences in solution conditions. Brain natriuretic peptide (BNP) is a novel peptide originally isolated from porcine brain [l]. It consists of 26 amino acid residues with the sequence shown in Fig. 1. An internal disulfide bond exists between the cysteine residues at positions 4 and 20, which gives rise to the expectation that this relatively small peptide may have a well-defined structure in solution.BNP closely resembles and elicits pharmacological responses very similar to atrial natriuretic peptide (ANP). These effects include natriuretic/diuretic, hypotensive and relaxant responses. It has therefore been suggested [l] that physiological functions such as water intake and salt appetite, which have previously been thought to be controlled by ANP, may also be regulated by BNP. BNP has been found to have a stronger inhibitory effect on cardiac function than ANP and like ANP, may play an important role in the pathogenesis of cardiovascular diseases [2]. Autoradiographic studies [3] have shown that BNP and ANP share binding sites in the heart and kidney. Radioimmunoassay studies [4] indicate that BNP is also synthesized in the heart and secreted from there into the circulatory system in a manner similar to ANP.A 2D NMR study [5] of a-human ANP concluded that it had a defined conformation in (C2H&30, although convergence amongst the six best conformers was not good, with a root-mean-square distance of 0.47 nm between superimposed backbone atoms. NMR studies [6 -91 of other closely related ANP analogues found no well-defined structures in solution.In view of the physiological importance of this recently discovered peptide, we have undertaken a 'H-NMR study of Abbreviations. BNP, brain natriuretic peptide; pBNP, porcine BNP; ANP, atrial natriuretic peptide; DQF-COSY, double-quantumfiltered proton-correlated spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; HOHAHA, homonuclear HartmannHahn spectroscopy; 2D, two dimensional. pBNP in (C2H3)2S0 using 2D techniques. While this manuscript was in preparation, a report [lo] appeared on the NMR assignment and conformation of pBNP in (C2H&SO-There are a number of assignments in that study which are markedly different to our own and these differences will be discussed in detail in the results section. ...

Solution structure of a defensin-like peptide from platypus venom

“…All cysteine residues found to be topologically equivalent by the DALI algorithm were disulphide-bonded in an identical fashion in each molecule. The anti-parallel β-strands in DLP-1 (15)(16)(17)(18)(37)(38)(39)(40) correspond closely with those in β-defensin-12 (10)(11)(12)(13)(32)(33)(34)(35)(36), although one of the strands in the latter is longer by one residue. Part of the large loop in DLP-1, defined by residues 24-27 and 30-36, corresponds to the region in β-defensin-12 defined by residues 21-31, which contains a β-strand and a βbulge.…”

“…The 4.2-kDa peptide, which shares a high degree of sequence identity with human and porcine C-type natriuretic peptide, has relaxant, oedema-producing and mast-cell-degranulating activities [11]. NMR studies of similar natriuretic peptides from various mammals revealed their tertiary structures to be flexible with little indication of well-defined secondary structure [12][13][14]. The other three protein components discovered in platypus venom do not resemble any of the proteins listed in the SwissProt database.…”

Structural Determinants of Natriuretic Peptide Receptor Specificity and Degeneracy

“…Previous NMR studies of free NP hormones show that they exist in an unstructured form unless rigidified synthetically. 24,25 The implications are that strategies to design mimics of small peptide hormones that exist as highly flexible ensembles of conformations in solution, based on calculations of low energy states, are inherently compromised by the fact that the receptor bound conformation need not be a stable or highly populated state in the unbound conformation. 25 It appears that in the case of the NP, hormone binding is inducing structure in the peptides.…”

Solution structure of a defensin-like peptide from platypus venom

“…All cysteine residues found to be topologically equivalent by the DALI algorithm were disulphide-bonded in an identical fashion in each molecule. The anti-parallel β-strands in DLP-1 (15)(16)(17)(18)(37)(38)(39)(40) correspond closely with those in β-defensin-12 (10)(11)(12)(13)(32)(33)(34)(35)(36), although one of the strands in the latter is longer by one residue. Part of the large loop in DLP-1, defined by residues 24-27 and 30-36, corresponds to the region in β-defensin-12 defined by residues 21-31, which contains a β-strand and a βbulge.…”