Lysine residues control the conformational dynamics of beta 2-glycoprotein I

Abstract

One of the major problems in the study of the dynamics of proteins is the visualization of changing conformations that are important for processes ranging from enzyme catalysis to signaling. A protein exhibiting conformational dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which exists in two conformations: the closed (circular) form and the open (linear) form. It is hypothesized that an increased proportion of the open conformation leads to the autoimmune disease antiphospholipid syndrome (APS). A characteristic feature of beta2GPI is the high content of lysine residues. However, the potential role of lysine in the conformational dynamics of beta2GPI has been poorly investigated. Here, we report on a strategy to permanently open up the closed protein conformation by chemical acetylation of lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific and complete acetylation was demonstrated by the quantification of primary amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as western blot analysis with an anti-acetylated lysine antibody. Our results demonstrate that acetylated beta2GPI preserves its secondary and tertiary structures, as shown by circular dichroism spectroscopy. We found that after lysine acetylation, the majority of proteins are in the open conformation as revealed by atomic force microscopy high-resolution images. Using this strategy, we proved that the electrostatic interaction of lysine residues plays a major role in stabilizing the beta2GPI closed conformation, as confirmed by lysine charge distribution calculations. We foresee that our approach will be applied to other lysine-rich proteins (e.g. histones) undergoing conformational transitions. For instance, conformational dynamics can be triggered by environmental conditions (e.g. pH, ion concentration, post-translational modifications, and binding of ligands). Therefore, our study may be relevant for investigating the equilibrium of protein conformations causing diseases.

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

“…Similarly, the DNA binding protein TCF4 is suggested to change conformations when acetylated in a complex with DNA [73], and Beta 2-glycoprotein changes from a closed to open conformation upon acetylation [74].…”

Atomic Force Microscopy Meets Biophysics, Bioengineering, Chemistry, and Materials Science

“…[50] The authors, by combining AFM with circulard ichroism (CD) spectroscopya nd dynamic light scattering, investigated the blood protein beta 2-glycoprotein, which presents two main conformations (opena nd closed). [50] The authors, by combining AFM with circulard ichroism (CD) spectroscopya nd dynamic light scattering, investigated the blood protein beta 2-glycoprotein, which presents two main conformations (opena nd closed).…”

“…Ar ecent example of the strength of AFM, in terms of high resolution, has been reported by Buchholz and co-workers. [50] The authors, by combining AFM with circulard ichroism (CD) spectroscopya nd dynamic light scattering, investigated the blood protein beta 2-glycoprotein, which presents two main conformations (opena nd closed). AFM images revealed that lysine acetylation promoted al arger population of proteins in an open conformation,w hich playedarole in the autoimmune disease antiphospholipids yndrome.…”

X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

“…The position of the O- and N-linked glycosylations is shown as triangles (▲) and stars (*), respectively. (B) Based on previous studies, β 2 GPI is believed to adopt an O-circular(27, 28), an S-twisted(26) and a J-elongated conformation(24, 25). The J-open conformation results upon interaction of DV with the phospholipids exposing the cryptic epitope R39-R43 (purple) to the solvent.…”

“…Importantly this epitope was also proposed to be cryptic based on the evidence that anti-DI antibodies showed no reactivity against β 2 GPI in solution but did react well when β 2 GPI was immobilized onto hydrophilic surfaces or plastic plates pre-coated with negatively charged phospholipids(18, 23). Second, using X-ray crystallography(24, 25), small-angle X-ray scattering (SAXS)(26), negative stain electron microscopy (EM)(27), and atomic force microscopy (AFM)(28), it was found that β 2 GPI can adopt O-circular, S-twisted and J-elongated conformations according to its ligation status. (Fig.…”