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

Abstract

Edited by Norma AllewellChemical 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 "overlength" 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.Present-day structural biology largely focuses on the predominant ground-state structures of proteins, which are most populated and readily detectable. Nevertheless, it is becoming clear that a protein can transiently adopt alternative, often lowly populated excited-state conformations. Dynamic interconversion between protein ground and excited states, together constituting the ensemble structures of a protein, enables the protein to perform its function (1, 2). For a multidomain protein, the dynamics usually involve the rearrangement between the domains, which can be essential for ligand recognition, catalytic activity, and allosteric modulation of the protein (3, 4). Despite technical advances in X-ray crystallography (5), nuclear magnetic resonance (NMR) spectroscopy (6), and cryo-electron microscopy (7), the excited states of proteins remain difficult to characterize. Among the existing techniques, NMR spectroscopy is known for identifying the excited states and for elucidating protein dynamics (6). Yet NMR requires a large quantity of purified, isotopically enriched recombinant proteins and is mainly applicable to proteins Ͻ50,000 Da.Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) 4 is an emerging technique in structural biology and has been increasingly used for modeling protein structures (8, 9). The cross-linked residues that are identified by high resolution mass spectroscopy should be close to each other, within the reach of the cross-linker used. However, it has been shown that the straight-line distance between t...

Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology

“…Thus, CLMS studies often produce high-confidence cross-links that cannot be explained by the available crystal structures used to benchmark them. Some of these discrepancies may represent conformations that are present in solution but are not available in the PDB 42 . Since cross-linking data can represent a mixture of all of the conformations occuring in the system, a careful analysis of long-distance crosslinks can be used to separate these alternative conformations.…”

Cross‐linked peptide identification: A computational forest of algorithms

“…The cross‐link might originate from an alternative conformation of the protein. DynaXL assesses conformational fluctuations of multidomain proteins. It calculates possible alternative conformations based on identified cross‐links and rigid body/torsion angle refinement and as such uses the supposed wrong distances to optimize the possible conformation of the protein complex.…”

ThreaDomEx: a unified platform for predicting continuous and discontinuous protein domains by multiple-threading and segment assembly

“…DLP-SVM ( 21 ), H-DROP ( 23 ) and DROP ( 22 ) provide domain linker positions and probability of the predictions based on SVM training. Finally, the ThreaDom server ( 30 ) uses multiple threading alignments and domain conservation score profiles to generate domain boundary assignments, which have witnessed successful applications in various 3D structure predictions ( 35 – 37 ) and structure-based function annotation studies ( 38 – 41 ).…”