Hydrogen/Deuterium Exchange Reflects Binding of Human Centrin 2 to Ca(2+) and Xeroderma Pigmentosum Group C Peptide: An Example of EX1 Kinetics

Abstract

Xeroderma pigmentosum (XP) is a genetic disease affecting 1 in 10,000-100,000 and predisposes people to early-age skin cancer, a disease that is increasing. Those with XP have decreased ability to repair UV-induced DNA damage, leading to increased susceptibility of cancerous non-melanomas and melanomas. A vital, heterotrimeric protein complex is linked to the nucleotide excision repair pathway for the damaged DNA. The complex consists of XPC protein, human centrin 2, and RAD23B. One of the members, human centrin 2, is a ubiquitous, acidic, Ca(2+)-binding protein belonging to the calmodulin superfamily. The XPC protein contains a sequence motif specific for binding to human centrin 2. We report here the Ca(2+)-binding properties of human centrin 2 and its interaction with the XPC peptide motif. We utilized a region-specific H/D exchange protocol to localize the interaction of the XPC peptide with the C-terminal domain of centrin, the binding of which is different than that of calmodulin complexes. The binding dynamics of human centrin 2 to the XPC peptide in the absence and presence of Ca(2+) are revealed by the observation of EX1 H/D exchange regime, indicating that a locally unfolded population exists in solution and undergoes fast H/D exchange.

Keywords: EX1 kinetics; centrin 2; hydrogen/deuterium exchange; protein; protein/peptide interaction; xeroderma pigmentosum.

Figure 1

The full length human centrin 2 sequence (top) compared to the delta-25 sequence (bottom). The amino acids in red were part of the start sequence in the expression vector. The amino acids in blue are those 25 which were deleted to make delta-25 human centrin 2. The numbering scheme refers to the full length human centrin 2 sequence.

Figure 2

Global H/D exchange kinetics experiments: Δ25-HC2 with no Ca²⁺ (1 mM EGTA) (squares), Δ25-HC2 with 2 mM Ca²⁺ (diamonds), 3:1 XPC:Δ25-HC2 with no Ca²⁺ (1 mM EGTA) (triangles), and 3:1 XPC:Δ25-HC2 with 2 mM Ca²⁺ (circles). H/D exchange was conducted over a 60 min time course with 97% D₂O, 10 mM HEPES (pH 7.4), 150 mM KCl, and 2.9 ! M Δ25-HC2. There is no fit curve at 3 min of exchange for the 3:1 complex in the absence of Ca²⁺ because there are two protein populations (see Figure 3). Error bars represent one standard deviation in the triplicate experiments.

Figure 3

The presence of two distinct protein populations at 3 min of H/D exchange. The lower mass species, representing bound protein, has an average deuterium uptake of 95 ± 3 Da. The higher mass species, representing unbound protein, has an average deuterium uptake of 122 ± 2. The spectrum is from one trial.

Figure 4

Region-specific H/D exchange kinetics of the N-terminus of Δ25-HC2, showing no differences in deuterium uptake and indicating that the differences observed for the intact protein occur in the C-terminal domain. Four states of Δ25-HC2 are shown: with no Ca²⁺ (squares), with 2 mM Ca²⁺ (diamonds), at 3:1 XPC:Δ25-HC2 and no Ca²⁺ (triangles), and at 3:1 XPC:Δ25-HC2 with 2 mM Ca²⁺ (circles).

Figure 5

Region-specific H/D exchange kinetics experiments of the C-terminus of Δ25-HC2. Peptide 110-136, part of EF-hand 3, exhibits EX1 exchange, revealed by two deuterium distributions at 3 min of exchange (see Figure 6). Peptide 146-162, part of EF-hand 4, becomes protected upon binding XPC in the absence and presence of Ca²⁺. There are small differences in deuterium uptake for the final C-terminal peptide, 163-172. Four states of Δ25-HC2 are shown: no Ca²⁺ (squares), with 2 mM Ca²⁺ (diamonds), 3:1 XPC:Δ25-HC2 and no Ca²⁺ (triangles), and 3:1 XPC:Δ25-HC2 with 2 mM Ca²⁺ (circles).

Figure 6

ESI mass spectra of peptide 110-136 ([M + 4H⁺] monoisotopic ion of m/z 786.19) at all H/D exchange times and in four binding states. The EX1 exchange regime is seen in panel 3, where low and high mass populations exist. Four states of Δ25-HC2 are shown: no Ca²⁺ (Panel 1), with 2 mM Ca²⁺ (Panel 2), 3:1 XPC:Δ25-HC2 and no Ca²⁺ (Panel 3), and 3:1 XPC:Δ25-HC2 with 2 mM Ca²⁺ (Panel 4). The dashed lines are for visual reference.

Figure 7

X-ray crystal structure (PDB: 2GGM) of human centrin 2 (red) in complex with Ca²⁺ (yellow) and the XPC peptide (blue). The XPC peptide interacts on the C-terminal tail of centrin, in agreement with the region-specific H/D exchange results. Peptide 110-136 of human centrin 2 is highlighted in green, 146-162 in orange, and 163-172 in purple.