Structural studies of membrane-associated complexes by solid-state NMR

Clay, Mary

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Description

Title

Structural studies of membrane-associated complexes by solid-state NMR

Author(s)

Clay, Mary

Issue Date

2015-01-21

Director of Research (if dissertation) or Advisor (if thesis)

Rienstra, Chad M.

Doctoral Committee Chair(s)

Rienstra, Chad M.

Committee Member(s)

• Oldfield, Eric
• Morrissey, James H.
• Tajkhorshid, Emad

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• solid-state nuclear magnetic resonance (NMR)
• Amphotericin B
• Ergosterol
• phosphatidylserine
• blood clotting

Abstract

The plasma membrane is one of the most important barriers in the human body. The membrane is far more than an inert platform supporting membrane proteins and their interactions. It is a complex mixture of lipids, sterols, and proteins, the exact composition of which directly regulates all extracellular interactions and trafficking of materials. The inherent asymmetry of the plasma membrane composition is directly involved in cell signaling processes and normal biological functions of the cell. This thesis utilizes solid-state nuclear magnetic resonance spectroscopy to investigate the structure of calcium-phosphatidylserine (PS) complexes that regulate blood coagulation, and to understand how interactions of the antifungal drug amphotericin B with ergosterol are related to its toxicity. The enzymatic activity of blood coagulation proteins is critically dependent upon the exposure of phosphatidylserine (PS) on the plasma membrane. Exposure of PS to plasma concentrations of calcium results in the formation of PS-rich nanodomains on the membrane surface, which are proposed as high affinity binding sites for coagulation factors. Phosphatidylethanolamine (PE) has been shown to enhance the pro-coagulation activity of PS membranes, and is hypothesized to play a role in the formation of PS-rich nanodomains. Despite extensive research, there is still no atomic-resolution description of these PS-rich membrane-binding sites or explanation for how they regulate blood coagulation reactions on the membrane surface. We propose the “Anything But Choline” hypothesis: GLA domain binding to lipid bilayers is facilitated by a combination of ca. one PS-specific interaction and ca. five or six phosphate-specific interactions. The PS-specific interaction can only be satisfied by L-serine, while phosphate-specific interactions can be satisfied by any phospholipid with an accessible phosphate group, with the exception of PC, because the choline headgroup sterically hinders GLA domain access to its phosphate group. In this study, we used a combination of magic-angle spinning solid-state NMR (MAS SSNMR) and novel 13C,15N-isotopic labeling of phospholipid headgroups to develop an atomic resolution description of the structure and dynamics of PS-rich nanodomains and their interaction with GLA domains. Amphotericin B is a highly effective antimicrobial agent that has been used for over fifty years to treat life threatening systemic fungal infections with minimal development of microbial resistance, an increasingly critical feature with the rise of antimicrobial resistant pathogens. The widely accepted mechanism of AmB cytotoxicity has been the formation of ion channels in the plasma membrane. However, recent studies have shown that channel formation is not essential for cell killing. Rather, it has been shown that cell death occurs from the binding of ergosterol (Erg, Fig. 1b). Erg is the primary sterol found in fungi and regulates many key cellular functions. In this thesis, I examined AmB interactions with Erg using magic-angle spinning solid-state NMR techniques, including paramagnetic relaxation enhancement (PRE), 1H spin diffusion, order parameter determination by 1H-13C recoupling, and homonuclear 13C-13C correlation spectroscopy (DARR, hChhC and SPC5). Collectively, the NMR, functional, and other biophysical data have led to the development of a new model for toxicity: the AmB sterol sponge. In this model, we propose that AmB toxicity is the result of the physical extraction of Erg from the cell membrane, thus disrupting all Erg-dependent cellular processes.

Graduation Semester

2014-12

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Copyright 2014 Mary Clay

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