Resonance Assignment and Three-Dimensional Structure Determination of a Human α-Defensin, HNP-1, by Solid-State NMR

Abstract

Human α-defensins (HNPs) are immune defense mini-proteins that act by disrupting microbial cell membranes. Elucidating the three-dimensional structures of HNPs in lipid membranes is important for understanding their mechanisms of action. Using solid-state NMR, we have determined the threedimensional structure of HNP-1 in a microcrystalline state outside the lipid membrane, which provides benchmarks for structure determination and comparison with the membrane-bound state. From a suite of 2D and 3D magic-angle spinning experiments, 13 C and 15 N chemical shifts were obtained that yielded torsion angle constraints while inter-residue distances were obtained to restrain the three-dimensional fold. Together, these constraints led to the first high-resolution SSNMR structure of a human defensin. The SSNMR structure has close similarity to the crystal structures of the HNP family, with the exception of the loop region between the first and second β-strands. The difference, which is partially validated by direct torsion angle measurements of selected loop residues, suggests possible conformational variation and flexibility of this segment of the protein, which may regulate HNP interaction with the phospholipid membrane of microbial cells.

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

“…The NMR structure not only showed the same disulfide-constrained fold as the crystal structures but also indicated differences from HNP-3 86 in the conformation of the rigid loop connecting the first and second b-strands. 22 This loop conformation is also variable among different defensin crystal structures, suggesting it may be caused by sequence differences among a-defensins. 22 On reconstitution into DMPC/DMPG bilayers, HNP-1 exhibited much broader line widths, indicating lipid-induced conformational heterogeneity.…”

“…22 This loop conformation is also variable among different defensin crystal structures, suggesting it may be caused by sequence differences among a-defensins. 22 On reconstitution into DMPC/DMPG bilayers, HNP-1 exhibited much broader line widths, indicating lipid-induced conformational heterogeneity. But the peak positions remain largely unchanged from the microcrystalline state, indicating that the average conformation of the protein is unperturbed by lipid binding, which is expected for this disulfide-stabilized protein.…”

“…20,21 This full-structure determination approach has been applied to a human a-defensin, HNP-1. 22,23 The monomer 3D structure forms the basis for determining the oligomeric structure of membrane proteins. To constrain oligomeric packing, one needs to have intermolecular distances well beyond the typical range of 5 Å for NMR.…”

“…22 Experiments on microcrystalline, lipid-free, HNP-1 were conducted to resolve and assign all protein signals. Two-dimensional and three-dimensional correlation experiments, now standard in MAS NMR, 90,91 allowed the full assignment of HNP-1 resonances.…”

The Antimicrobial and Antiviral Applications of Cell-Penetrating Peptides

“…Two examples of AMPs in the recent studies include protegrin-1 (PG-1) [ 26 ] and human neutrophil peptide-1 (HNP-1) [ 27 ]. PG-1 is representative of many β-sheet AMPs in its disulfi de-linked structure and has an Arg-rich sequence.…”