High resolution crystal structure of substrate-free human neprilysin
Introduction
Neprilysin (NEP, EC 3.4.24.11), also known as neutral endopeptidase, neuropeptidase, CD10 and enkephalin, is a transmembrane protease that belongs to the M13 family of zinc metalloproteases. NEP can digest a broad range of peptide substrates including insulin, enkephalin, substance P, bradykinin, endothelin-1, neurotensin and amyloid-β (Kerr and Kenny, 1974, Skidgel et al., 1984, Howell et al., 1995). In addition to the broad substrate specificity, NEP can cleave each substrate at a number of locations but exhibits a preference for the amino side of hydrophobic residues (Hersh and Morihara, 1986).
NEP is located at the plasma membrane and consist of three domains. The short intracellular and transmembrane domains are 27 and 23 residues in length and the third larger extracellular catalytic domain which is 699 residues in length. The extracellular domain contains a large central cavity where the conserved zinc binding motif HEXXH is located. The two histidine residues are responsible for the coordination of the zinc ion, while the glutamate is directly involved in catalysis. This conserved region has been targeted for inhibition to produce an antihypertensive response. To date eight inhibitor bound structures of the human NEP extracellular domain are available in the Protein Data Bank (Oefner et al., 2000, Oefner et al., 2004, Sahli et al., 2005, Oefner et al., 2007, Glossop et al., 2011, Schiering et al., 2016) but a structure free of bound inhibitor or substrate has yet to be reported. Here, we report the first crystal structure of the substrate-free extracellular domain of human NEP.
Section snippets
Expression and purification
All reagents for expression and purification were sourced from Sigma-Aldrich or Fisher Scientific unless otherwise specified.
A clone of Pichia pastoris GS115 expressing the extracellular catalytic domain of human NEP was purchased from Invitrogen. The clone contained human NEP (Tyr51-Trp749) integrated into the P. pastoris genome. The cytoplasmic (Gly1-Glu27) and transmembrane (Ile28-Ala50) domains of NEP were absent in the construct to increase the probability of protein crystallisation. The
Results and discussion
Here we report the first substrate-free crystal structure for the extracellular catalytic domain of human NEP (Gly54-Trp749). The structure was determined in the trigonal space group P3221 with a single molecule in the asymmetric unit to a resolution of 1.9 Å. The protein used for this structural study was expressed and purified in P. pastoris and has been shown to be catalytically active (Fig. 1).
The reported structure was refined to give final Rwork and Rfree values of 18.99% and 23.54%
Conclusion
In this report we have presented a substrate-free crystal structure for the extracellular domain of human NEP. The active site of this structure has been compared to other available inhibitor bound structures revealing flexibility in active site residues W693, F106 and R110. Flexibility in these residues is critical for allowing the active site to accommodate a variety of substrates binding at a range of positions. Additionally, we also note the central cavity of NEP has striking electrostatic
Acknowledgements
We thank Diamond Light Source for access to beamline I03 (proposal mx17212) that resulted in the data presented here. S.M is supported by a post-graduate studentship from the Alzheimer’s Society (UK) awarded to K.R.A. and V.S. [grant number – 286 (AS-PhD2015b-006)]. This research made use of the Balena High Performance Computing (HPC) Service at the University of Bath – UK.
Author contributions
S.M performed all the experiments, analysed the data and wrote the manuscript. V.S. supervised the study and edited the manuscript. K.R.A. conceptualised and supervised the study, analysed the data and edited the manuscript.
Additional information
Accession code: The atomic coordinates and structure factors (code 6GID) have been deposited in the Protein Data Bank (http://www.pdb.org).
Competing financial interests
The authors declare no competing financial interests.
References (34)
- et al.
Characterisation of neprilysin (EC 3.4.24.11) S2′ subsite
FEBS Lett.
(1997) - et al.
Synthesis and evaluation of heteroarylalanine diacids as potent and selective neutral endopeptidase inhibitors
Bioorg. Med. Chem. Lett.
(2011) - et al.
Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (Enkephalinase) to the bacterial neutral endopeptidase Thermolysin
J. Biol. Chem.
(1986) - et al.
Neutral endopeptidase can hydrolyze β-amyloid (1–40) but shows no effect on β-amyloid precursor protein metabolism
Peptides
(1995) - et al.
Structure of substrate-free human insulin-degrading enzyme (IDE) and biophysical analysis of ATP-induced conformational switch of IDE
J. Biol. Chem.
(2007) - et al.
Structure of human neutral endopeptidase (neprilysin) complexed with phosphoramidon
J. Mol. Biol.
(2000) - et al.
Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase
Peptides
(1984) - et al.
Phenix: a comprehensive Python-based system for macromolecular structure solution
Acta Crystallogr. Sect. D Biol. Crystallogr.
(2010) - et al.
Electrostatics of nanosystems: application to microtubules and the ribosome
Proc. Natl. Acad. Sci.
(2001) - et al.
Announcing the worldwide Protein Data Bank
Nat. Struct. Mol. Biol.
(2003)
CASTp: computed atlas of surface topography of proteins
Nucleic Acids Res.
MolProbity: all-atom structure validation for macromolecular crystallography
Acta Crystallogr. Sect. D Biol. Crystallogr.
Features and development of Coot
Acta Crystallogr. Sect. D Biol. Crystallogr.
Locating interaction sites on proteins: the crystal structure of thermolysin soaked in 2% to 100% isopropanol
Proteins
How good are my data and what is the resolution?
Acta Crystallogr. Sect. D Biol. Crystallogr.
The purification and specificity of a neutral endopeptidase from rabbit kidney brush border
Biochem. J.
Amyloid-β degrading cryptidases: insulin degrading enzyme, presequence peptidase, and neprilysin
Cell. Mol. Life Sci.
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