Specific metallo-protein interactions and antimicrobial activity in Histatin-5, an intrinsically disordered salivary peptide

Abstract

Histatin-5 (Hst-5) is an antimicrobial, salivary protein that is involved in the host defense system. Hst-5 has been proposed to bind functionally relevant zinc and copper but presents challenges in structural studies due to its disordered conformation in aqueous solution. Here, we used circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy to define metallo-Hst-5 interactions in aqueous solution. A zinc-containing Hst-5 sample exhibits shifted Raman bands, relative to bands observed in the absence of zinc. Based on comparison to model compounds and to a family of designed, zinc-binding beta hairpins, the alterations in the Hst-5 UVRR spectrum are attributed to zinc coordination by imidazole side chains. Zinc addition also shifted a tyrosine aromatic ring UVRR band through an electrostatic interaction. Copper addition did not have these effects. A sequence variant, H18A/H19A, was employed; this mutant has less potent antifungal activity, when compared to Hst-5. Zinc addition had only a small effect on the thermal stability of this mutant. Interestingly, both zinc and copper addition shifted histidine UVRR bands in a manner diagnostic for metal coordination. Results obtained with a K13E/R22G mutant were similar to those obtained with wildtype. These experiments show that H18 and H19 contribute to a zinc binding site. In the H18A/H19A mutant the specificity of the copper/zinc binding sites is lost. The experiments implicate specific zinc binding to be important in the antimicrobial activity of Hst-5.

Zinc Binding by Histatin 5 Promotes Fungicidal Membrane Disruption in C. albicans and C. glabrata

“…The full mechanism of action remains unclear, but multiple cellular effects are known, including efflux of small molecules like potassium, magnesium [ 5 ], ATP [ 6 ], ROS generation [ 7 ], and cell cycle arrest [ 8 ]. At the same time, it was discovered that Hst 5 binds to multiple divalent cations including copper and zinc [ 9 ], potentially with physiological relevance [ 10 ], though the function of this capability is still poorly understood [ 11 , 12 , 13 , 14 , 15 , 16 ].…”

“…The effects of Zn 2+ binding on the conformation, oligomerization state, and membrane interactions of the Hst 5 peptide have been described [ 11 , 14 , 15 ], but this has also raised questions about the practical application of Zn 2+ to alter or improve fungicidal activity. Though there are studies on the effect of both copper [ 12 ] and iron [ 16 ] on Hst 5 killing activity, comparatively little is known about the direct effect of Zn 2+ on Hst 5 efficacy or the mechanism involved.…”

“…Hst 5 is capable of catalyzing vesicle fusion in the presence of Zn 2+ [ 10 ], indicating direct binding with membrane phospholipids and the ability to affect membrane structure. Importantly, Zn 2+ binding stabilizes Hst 5 in bioactive conformations [ 11 ] and also promotes dimerization [ 14 ]. Peptide dimerization and aggregation have a complex relationship with AMP activity [ 32 , 33 ] but are highly potentiating for some pore-forming peptides [ 34 , 35 , 36 ], and pre-assembly of oligomeric peptide structures can speed up pore formation [ 34 , 37 , 38 ].…”

Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25

“…Therefore, oligomerization and aggregation can be compatible with peptide functionality. It is worth noting that the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide and the peptide hormone calcitonin are already on the market, whereas AMPs, such as LL-37, are in clinical trials [ 57 , 58 , 59 , 60 , 61 ]. In line with this, TCP-25 was found to oligomerize in a reversible manner, which is compatible with its observed efficacy in multiple in vitro and in vivo models [ 13 , 14 , 15 , 20 ].…”

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins

“…These peptides of 28 and 38 amino acids respectively are derived from a larger precursor and have a random coil structure [ 13 ]. This overlap between antimicrobial and metal binding activities is also seen in histatin-5, a histidine rich antimicrobial peptide in human saliva, which binds to Zn and Cu [ 14 , 15 , 16 ].…”