Charles R. Sanders

Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules

Introduction: Structure of Membrane-Associated Molecules by NMR High Resolution Solid State NMR Spectroscopy of Membrane Samples 2.1. Sampling spinning methods 2.2. Mechanical orientation of bilayers 2.3. Magnetic orientation of bilayers 2.3.1. Oriented phospholipid bilayers 2.3.2. Incorporation of membrane-associated molecules 2.3.3. Future development of magnetically orientable lipid media Experimental Considerations for Magnetically-Orientable Membrane Systems 3.1. Spectrometer requirements 3.2. Isotopic labeling 3.3. Strong coupling Anisotropic Spin Interactions: The Source of Orientation-Based Structural Data 4.1. Dipolar coupling 4.2. Quadrupolar coupling 4.3. Chemical shift anisotropy 4.4. Spin relaxation Determining Structure and Dynamics of Membrane-Bound Molecules 5.1. Structure and dynamics directly from experimental measurements 5.1.1. Order matrix analysis 5.1.2. Torsion angle analysis 5.2. NMR data as structural constraints in molecular modeling 5.3. Molecular dynamics simulations followed by back calculation of data Future Prospects Acknowledgements References 421 422 422 423 423 424 429 429 430 430 430 432 432 433 433 434 436 437 437 437 438 439 440 442

The Amyloid Precursor Protein Has a Flexible Transmembrane Domain and Binds Cholesterol

Insights into Amyloidogenesis The amyloid-β (Aβ) peptides associated with Alzheimers disease are generated by cleavage of the transmembrane C-terminal domain (C99) of the amyloid precursor protein by the enzyme γ-secretase. Barrett et al. (p. 1168) used nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy to show that C99 contains surface-associated N- and C-terminal helices and a flexibly curved transmembrane helix that is well suited to processive cleavage by γ-secretase. Elevated cholesterol levels have been found to increase Aβ generation. NMR titration together with mutagenesis revealed a binding site for cholesterol within C99 that included a motif previously implicated in protein oligomerization. The structure of the amyloid precursor protein transmembrane domain allows processive cleavage and cholesterol binding that may enhance cleavage. C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.

Bicelles: a model membrane system for all seasons?

The support of the National Science Foundation (MCB-9513357), National institutes of Health (GM47485), and the American Heart Association (through an Established Investigatorship to CS) is gratefully acknowledged. We also thank our many colleagues with whom we have discussed bicelles, including RR Vold, A Pines, J Losonczi, J Struppe, J Prestegard, and friends from the past and present Prestegard lab.

Substrate specificity of γ-secretase and other intramembrane proteases

Abstract.γ-Secretase is a promiscuous protease that cleaves bitopic membrane proteins within the lipid bilayer. Elucidating both the mechanistic basis of γ-secretase proteolysis and the precise factors regulating substrate identification is important because modulation of this biochemical degradative process can have important consequences in a physiological and pathophysiological context. Here, we briefly review such information for all major classes of intramembranously cleaving proteases (I-CLiPs), with an emphasis on γ-secretase, an I-CLiP closely linked to the etiology of Alzheimer’s disease. A large body of emerging data allows us to survey the substrates of γ-secretase to ascertain the conformational features that predispose a peptide to cleavage by this enigmatic protease. Because substrate specificity in vivo is closely linked to the relative subcellular compartmentalization of γ-secretase and its substrates, we also survey the voluminous body of literature concerning the traffic of γ-secretase and its most prominent substrate, the amyloid precursor protein.

Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase

Opening the Portico Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane phosphotransferases that function in prokaryotic-specific metabolic pathways. Van Horn et al. (p. 1726) determined the structure of the 40-kilodalton functional homotrimer of E. coli DAGK by solution nuclear magnetic resonance spectroscopy. Each monomer comprises three transmembrane helices. The third transmembrane helix from each subunit is domain-swapped to pack against the first and second transmembrane helices from an adjacent subunit. These three helices frame a portico-like membrane-submerged cavity that contains residues critical for activity in close proximity to residues critical for folding. The structure provides insight into the determinants of lipid substrate specificity and phosphotransferase activity. Mutations reveal the distribution of sequence changes that alter folding and affect function in a membrane-bound enzyme. Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK’s three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK’s lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.

Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO

Buffered mixtures of the detergent 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) and dimyristoylphosphatidylcholine (DMPC) orient in the presence of a strong magnetic field over a wide range of water contents (at least 65-85%) and CHAPSO:DMPC molar ratios (typically 1:10-1:3). 31P NMR studies show that the phospholipid in such mixtures is oriented with its director axis perpendicular to the magnetic field. 31P and 2H NMR results also suggest that the structure and dynamics of the DMPC molecules are similar to that of pure phospholipids existing in the liquid crystalline (L alpha) bilayer phase. The ability of 1:5 CHAPSO:DMPC samples to orient is highly tolerant of large changes in temperature, pH, and ionic strength, as well as to the addition of substantial amounts of charged amphiphiles or soluble protein. However, 2H NMR studies of deuterated beta-dodecyl melibiose (DD-MB) solubilized in the system indicate the head group conformation and/or dynamics of this glycolipid analogue is dependent upon the CHAPSO concentration. Despite the latter results, the orientational versatility of the system, together with the nondenaturing properties of CHAPSO, makes this system useful in spectroscopic studies of membrane-associated phenomena.

A Sulfilimine Bond Identified in Collagen IV

Toughing It Out in Membranes The formation of crosslinks in the triple-helical structure of collagen is crucial to its function. In the case of collagen IV, which provides structural integrity to the basement membrane of animal tissues, the chemical nature of the crosslinks has been difficult to pin down. Vanacore et al. (p. 1230) report a combination of high-resolution mass spectrometric and nuclear magnetic resonance studies that establish the crosslink between hydroxylysine-211 and methionine-93 is a sulfimine group (-S=N-). This type of bond appears to have evolved to withstand mechanical stress as animals evolved into more structurally complex organisms. An unusual sulfilimine bond provides a reinforcing cross-link of an extracellular matrix protein, collagen IV. Collagen IV networks are ancient proteins of basement membranes that underlie epithelia in metazoa from sponge to human. The networks provide structural integrity to tissues and serve as ligands for integrin cell-surface receptors. They are assembled by oligomerization of triple-helical protomers and are covalently crosslinked, a key reinforcement that stabilizes networks. We used Fourier-transform ion cyclotron resonance mass spectrometry and nuclear magnetic resonance spectroscopy to show that a sulfilimine bond (-S=N-) crosslinks hydroxylysine-211 and methionine-93 of adjoining protomers, a bond not previously found in biomolecules. This bond, the nitrogen analog of a sulfoxide, appears to have arisen at the divergence of sponge and cnidaria, an adaptation of the extracellular matrix in response to mechanical stress in metazoan evolution.

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.

Direct binding of cholesterol to the amyloid precursor protein: An important interaction in lipid-Alzheimer's disease relationships?

It is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimers disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appear to be a pro-amyloidogenic factor. The recent observation that the C-terminal transmembrane domain (C99, also known as the beta-C-terminal fragment, or beta-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimers-cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway-amyloid-beta and the intracellular domain of the APP (AICD)-may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies for AD.

French Swimwear for Membrane Proteins

Integral membrane proteins (IMPs) represent 20 ± 30% of all proteins and well over 50% of the targets for existing drugs. 2] Native IMPs are embedded in the lipid bilayers of biological membranes. The purification of IMPs requires that they first be rendered water soluble. Once solubilized, IMPs may be TMreconstituted∫ back into lipid bilayers or can be directly characterized in soluble form. For example, both 3D crystal growth and solution NMR spectroscopy require the use of solubilized IMPs. Traditionally, membrane proteins are maintained in soluble form by using detergents, which are able to dissolve lipid bilayers to form water-soluble complexes with both lipids and IMPs (Figure 1). Such complexes of detergent with protein and possibly