Protein Tertiary Structure by Crosslinking/Mass Spectrometry

The earliest structural studies on proteins using crosslinking/mass spectrometry aimed to elucidate their tertiary three-dimensional structure. Tertiary structure modeling using crosslinking fell out of favor for almost two decades because crosslink data were not informative to aid structure modeling. Two game-changing trends emerged: using short-range crosslinkers that capture relevant modeling information and high-density crosslinking. High-density crosslinking uses unspecific crosslinkers to dramatically increase crosslink numbers. In addition, computational structure modeling methods made significant progress in exploiting CLMS data. The combination of high-density crosslinking and computational structure modeling enables the elucidation of tertiary protein structure in native environments. This sidesteps the key limitation of today’s structure determination methods, which are unable (except for a few, specialized methods) to probe the structure of proteins in cell lysates or even intact cells. Observing the structures of proteins within the cell and tracking structural changes under different cellular conditions are the ultimate challenges for structural biology. This, however, requires an experimental technique that can generate sufficient data for structure determination and is applicable in the native environment of proteins. Crosslinking/mass spectrometry (CLMS) and protein structure determination have recently advanced to meet these requirements and crosslinking-driven de novo structure determination in native environments is now possible. In this opinion article, we highlight recent successes in the field of CLMS with protein structure modeling and challenges it still holds. Observing the structures of proteins within the cell and tracking structural changes under different cellular conditions are the ultimate challenges for structural biology. This, however, requires an experimental technique that can generate sufficient data for structure determination and is applicable in the native environment of proteins. Crosslinking/mass spectrometry (CLMS) and protein structure determination have recently advanced to meet these requirements and crosslinking-driven de novo structure determination in native environments is now possible. In this opinion article, we highlight recent successes in the field of CLMS with protein structure modeling and challenges it still holds. We can better understand the function of a protein on a molecular and mechanistic level by analyzing its structure. Structural information boosts our ability to engineer proteins, design drugs, and comprehend the molecular basis of life. Thus, researchers developed several scientific methods to determine protein structure; structure determination methods and solved structures have earned – at least in part – six Nobel prizes in the past twenty years (1997, 2003, 2006, 2009, 2012, and 2017 11997: Chemistry; structural studies on the ATP synthetase; 2003: Chemistry; structural studies on the potassium channel; 2006: Chemistry; structural studies on the eukaryotic transcription apparatus; 2009: Chemistry: structural studies on the ribosome; 2012: Chemistry: structural studies on G-protein-coupled receptors; 2017: Chemistry: development of cryo-electron microscopy for high-resolution structures.). The main methods for solving structures at atomic resolution are X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy. Despite their indisputable progress, each of these methods has specific limitations. X-ray crystallography relies on the ability of proteins to form crystals with specific properties. NMR can probe protein structure in solution, but is limited to small proteins. X-ray crystallography and NMR both require highly purified protein. Cryo-electron microscopy can resolve the structure of protein assemblies that are typically large (>200 kDa), homogenous, and rigid, and is increasingly able to resolve the structure of individual proteins in favorable cases [1Bai X. et al.How cryo-EM is revolutionizing structural biology.Trends Biochem. Sci. 2015; 40: 49-57Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar]. The three main structure determination techniques share the same general issues: they tend to study proteins in artificial environments and provide often only partial structures. These artificial environments do not resemble the cellular environments in which proteins function. Thus, the function we deduce from structures might be artifactual and divorced from biology. Researchers are, therefore, developing methods, including in-cell NMR [2Sakakibara D. et structure determination in by in-cell NMR PubMed Scopus Google et in-cell structure determination of proteins at PubMed Scopus Google and cryo-electron Cryo-electron can it the molecular of in atomic Full Text Full Text PDF PubMed Scopus Google to probe protein structures this in or even in the In this opinion article, we that the tertiary structure of proteins can be in native environments using crosslinking/mass spectrometry Thus, CLMS might the key limitation of structure determination We a to CLMS and computational structure modeling and the combination of the two methods enables researchers to generate tertiary structure to for a to CLMS and protein modeling. the of in CLMS studies on modeling protein et and native a combination for structural Sci. 2015; PubMed Scopus Google using challenges and Full Text Full Text PDF PubMed Scopus Google et and an to understand the structure and function of molecular Biochem. 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