HDX-ESI-MS reveals enhanced conformational dynamics of the amyloidogenic protein beta(2)-microglobulin upon release from the MHC-1

Abstract

The light chain of the major histocompatibility complex class 1 (MHC-1), the protein beta(2)-microglobulin (beta(2)m), has amyloidogenic properties that arise only upon its dissociation from the MHC-1. Here hydrogen/deuterium exchange electrospray ionization mass spectrometry (HDX-ESI-MS) has been used to compare the solution dynamics of beta(2)m in its MHC-1 bound state compared with those of beta(2)m as a free monomer. The capability of tandem mass spectrometry to dissociate the MHC-1 into its individual constituents in the gas phase following deuterium incorporation in solution has permitted the direct observation of the exchange properties of MHC-1 bound beta(2)m for the first time. The HDX-ESI-MS data show clearly that the H-->D exchange of MHC-1 bound beta(2)m follows EX2 kinetics and that about 20 protons remain protected from exchange after 17 days. Free from the MHC-1, monomeric beta(2)m exhibits significantly different HDX behavior, which encompasses both EX1 and EX2 kinetics. The EX2 kinetics indicate a tenfold increase in the rate of exchange compared with MHC-1 bound beta(2)m, with just 10 protons remaining protected from EX2 exchange and therefore exchanging only via the EX1 mechanism. The EX1 kinetics observed for unbound beta(2)m are consistent with unfolding of its exchange-protected core with a t(1/2) of 68 min (pH 7, 37 degrees C). Thus, upon dissociation from the stabilizing influence of the MHC-1, free beta(2)m becomes highly dynamic and undergoes unfolding transitions that result in an aggregation-competent protein.

Graphical abstract

Figure 1

Ribbon diagram of the MHC-1 (PDB 2VLL) [41] showing the heavy chain (α1, α2, α3; gray ribbon), the light chain (β₂m; black ribbon), and a peptide (black stick) that binds in the cleft formed by the two helical regions (α1 and α2) of the heavy chain. Drawn using PyMOL v.0.99 [42].

Figure 2

(a) ESI-MS m/z spectrum of the MHC-1 (pH 7.0), showing predominantly intact MHC-1 (C), together with traces of MHC-1 minus peptide (D), free β₂m (B), and peptide (A). (b) ESI-MS/MS m/z spectrum showing dissociation of intact MHC-1 (C; +13 charge state ions, m/z 3442) to yield: MHC-1 minus peptide (D), MHC-1 heavy chain (E), free β₂m (B), and peptide (A). The number immediately following the letters A, B, C, D, and E indicates the charge state of those ions.

Figure 3

Time course showing H→D exchange (pH 7.0 corrected, 37 °C) of β₂m, which has been dissociated from intact MHC-1 after HDX by ESI-MS/MS. The +5 charge state ions are highlighted showing m/z versus intensity over the time course. The centroided m/z values of the ions are indicated on each spectrum and the centroided m/z of the first time point is shown throughout as a dashed line. The unexchanged +5 charge state ions (t = 0) are shown in the inset.

Figure 4

The increase in molecular mass of β₂m observed by ESI-MS/MS dissociation of the intact MHC-1 over the course of H→D exchange (pH 7.0 corrected, 37 °C). A single-exponential fit (gray line) indicates a rate constant of 0.002 ± 0.0004 min⁻¹. The theoretical end point of the reaction (12,044 Da; dashed line) is shown; about 20 protons remain protected.

Figure 5

HDX-ESI-MS of unbound, monomeric β₂m (pH 7, 37 °C) highlighting the +6 charge state ions showing m/z versus intensity over the time course of the D→H exchange reaction. The black (higher m/z) peaks show the population of protein molecules in solution that are undergoing exchange by an EX2 mechanism alone, whereas the gray (lower m/z) peaks indicate the molecules in solution that have experienced exchange by the EX1 mechanism. The centroided m/z values of the ions are indicated on each spectrum and the centroided m/z of the first time point is shown throughout as a dashed line. The unexchanged +6 charge state ions (t = 0) are shown in the inset.

Figure 6

(a) The intensity of the EX1 peak relative to the total protein signal during the D→H exchange reaction monitored by HDX-ESI-MS of unbound β₂m (pH 7, 37 °C). The data fit to a single exponential (gray line) with t_1/2 = 67.6 ± 3 min. (b) The increase in mass of unbound β₂m undergoing HDX by an EX2 mechanism observed by ESI-MS over the course of H→D exchange (pH 7.0 corrected, 37 °C). A single-exponential fit (gray) indicates a rate of 0.023 ± 0.002 min⁻¹. The theoretical end point of the reaction (12,044 Da; dashed line) is shown; about 10 protons exchange only via an EX1 mechanism.