Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links

Abstract

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function.

Keywords: calmodulin (CaM); mass spectrometry (MS); molecular dynamics; protein cross-linking; protein domain; protein dynamics; protein excited state; structural model.

FIGURE 1.

Protein dynamics can be manifested from over-length cross-links, as illustrated here with the open-closed domain movement for a two-domain protein. A cross-linking reagent (e.g. BS³ or BS²G) first reacts with a lysine residue to form a mono-linked intermediate. The second conjugation reaction may only take place when the protein fluctuates to the alternative closed conformation.

FIGURE 2.

Schematic for characterizing protein ensemble structures based on CXMS data as implemented in DynaXL. The workflow of DynaXL includes four steps: CXMS analysis, preparation of the structure file, structure refinement with explicit modeling, and validation. Domain boundaries in multidomain proteins are delineated by multithreading alignment and validated with MD simulations, allowing the classification of intra- versus interdomain cross-link. The intradomain cross-links are examined against the known structures of the protein for compatibility so as to confirm the rigidity of each domain. Based on interdomain cross-links, protein ensemble structure was refined using conjoined rigid-body/torsion angle-simulated annealing. If a two-conformer ensemble structure cannot account for all interdomain cross-links, DynaXL attempts a three-conformer ensemble structure and so on until all cross-links are explained. Lastly, the alternative conformational states are subjected to further assessment and validation.

FIGURE 3.

Ligand-free Ca²⁺-CaM can fluctuate between open and closed states. A, the interdomain cross-links (indicated by red dotted lines) involve residues too far away to be cross-linked in the open state. Note that Lys²¹ and Lys⁹⁴ are each cross-linked with two different residues. For clarity, the N-hydroxysuccinimide ester at one end of the cross-linker and the Ca²⁺ ions bound to the protein are not shown. B, ensemble refinement with explicit modeling of the cross-linkers using the DynaXL approach revealed the closed-state structure of Ca²⁺-CaM, which would allow the cross-linking reaction to take place (indicated by red lines).

FIGURE 4.

Assessment of the closed-state structure of ligand-free Ca²⁺-CaM. A, comparison to the closed-state structure obtained by refining against the PRE NMR data. B, comparison to the closed-state structure of ligand-bound Ca²⁺-CaM, which was previously determined using X-ray crystallography (PDB code 2BE6).

FIGURE 5.

Visualizing the conformational fluctuations of EIN from over-length cross-links. A, the structure of EIN either by itself or in complex with HPr cannot explain the two cross-links between α-domain and α/β-domain (indicated by red dotted lines). B, modeled from interdomain cross-links, EIN is found to exist in an alternative conformation, likely responsible for phosphoryl receiving. This conformational state allows BS²G cross-link between Lys²⁰ and Lys⁴⁹ and the BS³ cross-link between Lys¹⁷⁵ and Lys⁴⁹ (indicated with red lines) to be formed.

FIGURE 6.

Visualizing the conformational fluctuations of apoQBP from MS-identified cross-links. A, the apoQBP exists in an open conformation and cannot account for the two interdomain cross-links (denoted by red dotted lines). B, with the DynaXL modeling, apoQBP is found to exist in a partially closed conformation, which enables the cross-linking reactions between Lys⁷⁶ and Lys¹²⁵ and between Lys⁷⁷ and Lys¹²⁵ (indicated with red lines).