MAPping the Chiral Inversion and Structural Transformation of a Metal-Tripeptide Complex Having Ni-Superoxide Dismutase Activity

Abstract

The metal abstraction peptide (MAP) tag is a tripeptide sequence capable of abstracting a metal ion from a chelator and binding it with extremely high affinity at neutral pH. Initial studies on the nickel-bound form of the complex demonstrate that the tripeptide asparagine-cysteine-cysteine (NCC) binds metal with 2N:2S, square planar geometry and behaves as both a structural and functional mimic of Ni superoxide dismutase (Ni-SOD). Electronic absorption, circular dichroism (CD), and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic NiII center. It is apparent from the CD signal of Ni-NCC that the optical activity of the complex changes over time. Mass spectrometry data show that the mass of the complex is unchanged. Combined with the CD data, this suggests that chiral rearrangement of the complex occurs. Following incubation of the nickel-containing peptide in D2O and back-exchange into H2O, incorporation of deuterium into non-exchangeable positions is observed, indicating chiral inversion occurs at two of the alpha carbon atoms in the peptide. Control peptides were used to further characterize the chirality of the final nickel-peptide complex, and DFT calculations were performed to validate the hypothesized position of the chiral inversions. In total, these data indicate Ni-SOD activity is increased proportionally to the degree of structural change in the complex over time, as cross-correlation between the change in CD signal and change in SOD activity reveals a linear relationship.

Mechanistic investigations of matrix metalloproteinase-8 inhibition by metal abstraction peptide

“…Metal binding to MAP requires neutral to basic pH. 28 Therefore, samples were subsequently rinsed with acetate buffers pH 3.5, pH 3.5 with EDTA, and pH 4.5 with EDTA, and then incubated in acetate buffer pH 4.5 with EDTA for several hours to disrupt MAP binding to Zn 2þ .…”

The Outer-Coordination Sphere: Incorporating Amino Acids and Peptides as Ligands for Homogeneous Catalysts to Mimic Enzyme Function

“…25, left), in this case using an Asn-Cys-Cys as an even smaller active site model (115). A follow-up study provided insight into a chiral inversion at two of the amino acids that is important as to how the catalyst mimic functions and may provide insight into the function of NiSOD (116).…”

Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli

“…27 The cla MP Tag is a tripeptide consisting of the amino acid sequence Asn-Cys-Cys, which scavenges small transition metals from chelating agents. 28 Because of the uniquely beneficial properties of the cla MP Tag, which includes extraordinarily tight binding, resistance to metal release upon dilution at serum pH and specific release in acidic conditions as in endosomes, the cla MP Tag is being investigated as a method for targeted delivery of metals for therapeutic and diagnostic applications by designing targeting proteins to contain the metal bound tripeptide. 27 The cla MP Tag has not been investigated previously in a system, such as matrix metalloproteinases, in which a structural and/or catalytic metalbinding site is present.…”