Fast cross-linking by DOPA2 promotes the capturing of a stereospecific protein complex over nonspecific encounter complexes

Abstract

Transient and weak protein-protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes EIN/HPr and EIIA^Glc/EIIB^Glc as our model systems, we evaluated the effects of two amine-specific homo-bifunctional cross-linkers with different reactivities. We showed previously that DOPA2 (di-ortho-phthalaldehyde with a di-ethylene glycol spacer arm) cross-links proteins 60-120 times faster than DSS (disuccinimidyl suberate). We found that though most of the intermolecular cross-links of either cross-linker are consistent with the encounter complexes (ECs), an ensemble of short-lived binding intermediates, more DOPA2 intermolecular cross-links could be assigned to the stereospecific complex (SC), the final lowest-energy conformational state for the two interacting proteins. Our finding suggests that faster cross-linking captures the SC more effectively and cross-linkers of different reactivities potentially probe protein-protein interaction dynamics across multiple timescales.

Keywords: Chemical cross-linking; Cross-linker; DOPA2; DSS; Encounter complexes; Mass spectrometry; Stereospecific complex; Transient protein–protein interaction.

Figure 1

Performance of DOPA2 and DSS on weak protein complexes. A The chemical structures of DOPA2 and DSS. B Conceptual diagram of carbohydrate transport and phosphorylation by the phosphotransferase system. C,D SDS-PAGE of DOPA2 or DSS-cross-linked EIN/HPr, EIIA^Glc/EIIB^Glc complexes, with the protein concentrations comparable to the respective equilibrium dissociation constant. Heterodimer bands with the expected molecular weights were marked by square brackets. Cross-linking of EIN/HPr with 0.8 mmol/L DOPA2 resulted in high Mw products that did not enter the separating gel

Figure 2

In comparison to DSS, DOPA2 captured more non-covalent dimers readily visible on SDS-PAGE. A,B SDS-PAGE of cross-linked EIN/HPr and EIIA^Glc/EIIB^Glc complexes. Captured protein heterodimers were marked by square brackets. C,D The relative intensity of the heterodimer band, normalized by the total intensity. E,F The inter-molecular or intra-molecular residue pairs identified in the covalent dimer bands cross-linked by DOPA2 or DSS. G,H DOPA2 cross-linked or DSS cross-linked residue pairs identified from excised dimer bands were mapped to the primary sequences of the respective complexes (visualized using xiNET (Combe et al. 2015)). Cross-links were filtered by requiring FDR < 0.01 at the spectra level, E-value < 1 × 10⁻³ and spectral counts ≥ 3

Figure 3

Contrasting cross-link identification results before and after SDS-PAGE purification. A Schematic diagram of in-gel digestion or in-solution digestion of EIN/HPr complex after DOPA2 or DSS cross-linking. B The inter-molecular or intra-molecular residue pairs identified in the EIN/HPr complex cross-linked by DOPA2 or DSS with purification by SDS-PAGE or not. The number of spectra identified is shown below. C,D The inter-molecular residue pairs identified by DOPA2 in the dimer band or in solution were mapped to the primary sequence of EIN/HPr (visualized using xiNET (Combe et al. 2015)). E,F As in panels C and D, but for cross-links identified by DSS. The stereospecific interface between EIN and HPr was indicated by light orange color. The green lines and the orange lines denoted the cross-links that were assigned to ECs and SC, respectively. The numbers of the corresponding spectra of each cross-link were indicated by the thickness of the line. Cross-links were filtered by requiring FDR < 0.01 at the spectra level, E-value < 1 × 10^-8 and spectral counts > 3

Figure 4

Systematic analysis of cross-linked protein species separated by SDS-PAGE. A SDS-PAGE of DOPA2 or DSS-cross-linked EIN/HPr and the demarcation range for systematic excision of the gel slides, arbitrarily denoted as L1 through L6. B,C The number of inter- or intra-molecular residue pairs identified for DOPA2 or DSS cross-linking in L1–L6, respectively. EIN(30)-EIN(30), the only unambiguously identified homodimeric cross-link suggestive of higher-order interactions between the EIN/HPr heterodimers, was included in this graph. It came from L1 and L2 of the DOPA2 treated sample or L1, L2 and L4 of the DSS treated sample. D,E The number of inter- or intra-molecular spectra identified for DOPA2 or DSS cross-linking in L1–L6, respectively. Cross-links were filtered by requiring FDR < 0.01 at the spectra level, E-value < 1 × 10^-8 and spectral counts in each sample ≥ 2

Figure 5

Analysis of the heterodimeric cross-links with respect to the conformations of the stereospecific and encounter complexes. A Three representative encounter structures (EC-I, EC-II, and EC-III) and stereospecific complex (SC, PDB code: 3EZA) (Fawzi et al. ; Garrett et al. ; Tang et al. 2006) for EIN/HPr complex. B The number of DOPA2 or DSS cross-linked inter-links of EIN/HPr compatible with SC and ECs w/t or w/o gel purification. C The relative percentages of DOPA2 or DSS cross-linked inter-links of EIN/HPr compatible with SC and ECs w/t or w/o gel purification. D The number of DOPA2 or DSS cross-linked spectra of EIN/HPr compatible with SC and ECs in L1–L3, respectively. E The relative percentages of DOPA2 or DSS cross-linked spectra of EIN/HPr compatible with SC and ECs in L1–L3, respectively. Cross-links were filtered by requiring FDR < 0.01 at the spectra level, E-value < 1 × 10⁻⁸ and spectral counts in each sample ≥ 2

Figure 6

Proposed model for the conformational preference by DOPA2 and DSS in cross-linking. (1) An EC is too transient for either DOPA2 or DSS to capture immediately. (2) Compared to DSS, the fast-reacting cross-linker DOPA2 has a higher chance of capturing the stereospecific complex of EIN/HPr, by forming an inter-molecular cross-link before the two subunits dissociate. (3) Cross-linking by DSS is too slow to capture an EIN/HPr complex in one shot. DSS cross-linking probably takes place in two steps: first a DSS mono-link forms, then in one or more subsequent dissociation/association cycles, the mono-link planted on a subunit turns into a cross-link. Between the mono-link and the cross-link, the protein has many opportunities to sample various alternative conformational states, including the ECs