Continuous and Pulsed Hydrogen–Deuterium Exchange and Mass Spectrometry Characterize CsgE Oligomerization

Abstract

We report the use of hydrogen-deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called Curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we first determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS we observed an interesting structural rearrangement that occurs during the oligomerization of CsgE.

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation

“…Cluster 2, especially, shows a highly exposed hydrophobic region. The clustered distribution of exposed hydrophobic residues in CsgE structure agrees with our previous study, using hydrogen-deuterium amide exchange coupled to mass spectrometry (41), showing regions 23-36, 39-51, and 67-79 are involved in CsgE self-association. The structural position of the exposed hydrophobic residues suggests the double mutant W48A/F79A can disrupt the main exposed hydrophobic cluster, resulting in weaker propensity to self-associate.…”

“…One primary barrier to structure elucidation has been the mixture of oligomeric species formed by purified CsgE. However, we have recently used molecular footprinting techniques to generate a double mutant of CsgE (W48A/F79A) that is more stable and monodispersed at high concentrations (41). As shown here, the behavior of this double-mutant CsgE appears to be similar to that of the WT protein in overall structure and function.…”

“…Wild-type (WT) CsgE tends to self-associate to oligomers in a concentration-and temperature-dependent manner. In our previous report, residues responsible for the oligomerization have been identified by hydrogen-deuterium amide exchange coupled with mass spectrometry, and a double-mutant CsgE W48A/F79A was found to undergo significantly less self-association and to be more stable than the WT protein (41). As illustrated in Fig.…”

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

“…By massively increasing its interest in protein therapeutics within these last years, the biopharmaceutical industry became particularly interested in improving and developing spatially resolved analytical techniques like HDX-MS for its needs in both research and quality control. (14,15) Since so far HDX-MS was mostly implemented on monomeric proteins (16)(17)(18)(19)(20)(21)(22) and only a few studies have been conducted on homo-oligomeric proteins (23)(24)(25)(26)(27) or protein complexes, (28)(29)(30)(31) efforts have to be continued for extracting the dynamic determinants of complexes.…”

Understanding curli amyloid-protein aggregation by hydrogen–deuterium exchange and mass spectrometry

“…24 Recently, we applied both continuous and pulsed HDX-MS studies to another curli family member, CsgE, to determine the regions involved in self-association, and we discovered a conformational rearrangement during the oligomerization process. 26 Extension of this platform to regulated CsgA aggregation provides an opportunity to understand region-specific aggregation for the near-native state. In addition, we complemented pulsed HDX-MS with transmission electron microscopy (TEM) to probe both soluble and insoluble CsgA proteins.…”