EMSL: David W Hoyt's Publications

Chen B, X Ren, T Neville, WG Jerome, DW Hoyt, DL Sparks, G Ren, and J Wang. 2009. "Apolipoprotein AI tertiary structures determine stability and phospholipid-binding activity of discoidal high-density lipoprotein particles of different sizes." Protein Science 18(5):921-935. doi:10.1002/pro.101 Abstract Human high-density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140-240 kDa, disk-shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high-resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5-10% lipid-poor apoAI that significantly interferes with the high-resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid-poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high-resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid-binding activity. Interestingly, these property/functional differences are independent from the apoAI -helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two-dimensional NMR and negative stain electron microscopy data. Our result further provides the first high-resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.

Hu JZ, JA Sears, Jr, JH Kwak, DW Hoyt, Y Wang, and CHF Peden. 2009. "An Isotropic Chemical Shift-Chemical Shift Anisotropic Correlation Experiment Using Discrete Magic Angle Turning." Journal of Magnetic Resonance 198(1):105-110. Abstract An isotropic-anisotropic shift 2D correlation spectroscopy is introduced that combines the advantages of both magic angle turning (MAT) and magic angle hopping (MAH) technologies. In this new approach, denoted DMAT for "discrete magic angle turning", the sample rotates clockwise followed by an anticlockwise rotation of exactly the same amount with each rotation less or equal than 360 degrees but greater than 240 degrees, with the rotation speed being constant only for times related to the evolution dimension. This back and forth rotation is repeated and synchronized with a special radio frequency (RF) pulse sequence to produce an isotropic-anisotropic shift 2D correlation spectrum. For any spin-interaction of rank-2 such as chemical shift anisotropy, isotropic magnetic susceptibility interaction, and residual homonuclear dipolar interaction in biological fluid samples, the projection along the isotropic dimension is a high resolution spectrum. Since a less than 360 degrees sample rotation is involved, the design potentially allows for in situ control over physical parameters such as pressure, flow conditions, feed compositions, and temperature so that true in-situ NMR investigations can be carried out.

Briknarova K, X Zhou, AC Satterthwait, DW Hoyt, KR Ely, and S Huang. 2008. "Structural Studies of the SET Domain from RIZ1 Tumor Suppressor." Biochemical and Biophysical Research Communications 366(3):807-813. doi:10.1016/j.bbrc.2007.12.034 Abstract Histone lysine methyltransferases (HKMTs) are involved in regulation of chromatin structure, and, as such, are important for longterm gene activation and repression that is associated with cell memory and establishment of cell-type specific transcriptional programs. Most HKMTs contain a SET domain, which is responsible for their catalytic activity. RIZ1 is a transcription regulator and tumor suppressor that catalyzes methylation of lysine 9 of histone H3 and contains a rather distinct SET domain. Similar SET domains, sometimes refererred to as PR (PRDI-BF1 and RIZ1 homology) domains, are also found in other proteins including Blimp-1/PRDI-BF1, MDS1-EVI1 and Meisetz. We determined the solution structure of the PR domain from RIZ1 and characterized its interaction with S-adenosyl homocysteine (SAH) and a peptide from histone H3. Despite low sequence identity with canonical SET domains, the PR domain displays a typical SET fold including a pseudo-knot at the C-terminus. The N-flanking sequence of RIZ1 PR domain adopts a novel conformation and interacts closely with the SET fold. The C-flanking sequence contains an α-helix that exhibits higher mobility than the SET fold and points away from the protein face that harbors active site in other SET domains. Residues that interact with the methylation cofactor in SET domains are not conserved in RIZ1 or other PR domains, and the SET fold of RIZ1 does not bind SAH. However, the PR domain of RIZ1 interacts specifically with a synthetic peptide comprising residues 1-20 of histone H3.

Ren X, Y Yang, T Neville, DW Hoyt, DL Sparks, and J Wang. 2007. "A complete backbone spectral assignment of human apolipoprotein AI on a 38 kDa preβHDL (Lp1-AI) particle ." Biomolecular NMR Assignments 1(1):69-71. doi:DOI 10.1007/S12104-007-9020-5 Abstract Apolipoprotein A-I (apoAI, 243-residues) is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. This important property of apoAI is related to its roles in reverse cholesterol transport pathway. Upon lipid-binding, apoAI undergoes conformational changes from lipid-free to several different HDL-associated states (1). These different conformational states regulate HDL formation, maturation and transportation. Two initial conformational states of apoAI are lipid-free apoAI and apoAI/preβHDL that recruit phospholipids and cholesterol to form HDL particles. In particular, lipid-free apoAI specifically binds to phospholipids to form lipid-poor apoAI, including apoAI/preβ-HDL (~37 kDa). As a unique class of lipid poor HDL, both in vitro and in vivo evidence demonstrates that apoAI/preβ-HDLs are the most effective acceptors specifically for free cholesterol in human plasma and serves as the precursor of HDL particles (2). Here we report a complete backbone spectral assignment of human apoAI/preβHDL. Secondary structure prediction using backbone NMR parameters indicates that apoAI/preβHDL displays a two-domain structure: the N-terminal four helix-bundle domain (residues 1-186) and the C-terminal flexible domain (residues 187-243). A structure of apoAI/preβ-HDL is the first lipid-associated structure of apoAI and is critical for us to understand how apoAI recruits cholesterol to initialize HDL formation. BMRB deposit with accession number: 15093.

Smirnov S, NG Isern, ZG Jiang, DW Hoyt, and CJ Mcknight. 2007. "The Isolated Sixth Gelsolin Repeat and Headpiece Domain of Villin Bundle F-Actin in the Presence of Calcium and Are Linked by a 40-Residue Unstructured Sequence ." Biochemistry 46(25):7488-7496. doi:10.1021/bi700110v Abstract Villin is an F-actin regulating, modular protein with a gelsolin-like core and a distinct C-terminal 'headpiece’ domain. Localized in the microvilli of the absorptive epithelium, villin can bundle F-actin and, at higher calcium concentration, is capable of a gelsolin-like F-actin severing. The headpiece domain can, in isolation, bind F-actin and is crucial for F-actin bundling by villin. While the three-dimensional structure of the isolated headpiece is known, its conformation in the context of attachment to the villin core remains unexplored. Furthermore, the dynamics of the linkage of headpiece to the core has not been determined. To address these issues, we employ a 208 residue modular fragment of villin, D6-HP, which consists of the sixth gelsolin-like domain of villin (D6) and the headpiece (HP). We demonstrate that this protein fragment requires calcium for structural stability and, surprisingly, is capable of Ca2+-dependent F-actin bundling, suggesting that D6 contains a cryptic F-actin binding site. NMR resonance assignments and 15N-relaxation measurements of D6-HP in 5 mM Ca2+ demonstrate that D6-HP consists of two independent structural domains (D6 and HP) connected by an unfolded 40-residue linker sequence. The headpiece domain in D6-HP retains its structure and interacts with D6 domain only through the linker sequence without engaging in other interactions. Chemical shift values indicate essentially the same secondary structure elements for the D6 domain in D6-HP as in the highly homologous gelsolin domain 6. Thus, the headpiece domain of villin is structurally and functionally independent from the core domain.

Yang Y, DW Hoyt, and J Wang. 2007. "A Complete NMR Spectral Assignment of the Lipid-free Mouse Apolipoprotein A-I (ApoAI) C-terminal Truncation Mutant, ApoAI(1-216)." Biomolecular NMR Assignments 1(1):109-111. doi:10.1007/s12104-007-9031-2 Abstract Apolipoprotein A-I (apoAI) is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. Upon lipid-binding, apoAI undergoes conformational changes from lipid-free to several different HDL-associated states (1). These different conformational states regulate HDL formation, maturation and transportation. Recent crystal structure of lipid-free human apoAI represents a major progress of structural study of lipid-free apoAI (2). However, no structural is available for lipid-free mouse apoAI (240-residues). Since mouse HDL is homogenous with only HDL2-like size, whereas human HDL is heterogeneous, containing HDL2/HDL3 as its main species, a structural comparison between human and mouse apoAI may allow us to identify structure basis of HDL size distribution difference between human and mouse. We carried out an NMR structure determination of lipid-free mouse apoAI (1-216) and completely assigned backbone atoms (except backbone amide proton and nitrogen atoms for residues D1, N48, W107, K108, K132, E135, F147, R148, M169 and K203). Secondary structure prediction using backbone NMR parameters indicates that lipid-free mouse apoAI consists of a four helical segments in the N-terminal domain, residues 1-180. In addition, two short helices are also observed between residues 190-195 and 210-215. The helix locations are significantly different from those in the crystal structure of human apoAI, suggesting that mouse apoAI may have a different conformational adaptation upon lipid-binding. BMRB deposit with accession number: 15091.

Brzovic P, AV Lissounov, D Christensen, DW Hoyt, and RE Klevit. 2006. "A UbcH5/Ubiquitin Noncovalent Complex is Required for Processive BRCA1-Directed Ubiquitination." Molecular Cell 21(6):873-880. Abstract Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved a/b-fold core domain ofw150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/ Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5cwUb molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1

Briknarova K, F Nasertorabi, ML Havert, E Eggleston, DW Hoyt, C Li, AJ Olson, K Vuori, and KR Ely. 2005. "The Serine-rich Domain from Crk-associated Substrate (p130Cas) is a Four-helix Bundle." Journal of Biological Chemistry 280(23):21908-21914. Abstract p130Cas (Crk-associated substrate) is a docking protein that is involved in assembly of focal adhesions and concomitant cellular signaling. It plays a role in physiological regulation of cell adhesion, migration, survival and proliferation, as well as in oncogenic transformation. The molecule consists of multiple protein-protein interaction motifs including a serine-rich region that is positioned between Crk and Src-binding sites. This study reports the first structure of a functional domain of Cas. The solution structure of the serine-rich region has been determined by nuclear magnetic resonance spectroscopy (NMR) demonstrating that this is a stable domain that folds as a four-helix bundle, a protein-interaction motif.

Xu C, A Sivashanmugam, DW Hoyt, and J Wang. 2005. "A Complete Backbone Assignment of the Apolipoprotein E LDL Receptor Binding Domain [Letter to the Editor]." Journal of Biomolecular NMR 32(2):177. doi:10.1007/s10858-005-6729-2 Abstract Human apolipoprotein E (apoE) is a 299-residue exchangeable apolipoprotein that was initially recognized as a major determinant in lipoprotein metabolism and cardiovascular diseases. Recent evidence has indicated that apoE also plays critical roles in several other important biological processes not directly related to its lipid transport function, including Alzheimer’s disease, cognitive function, immunoregulation, cell signaling, and possibly even infectious diseases. ApoE contains two structural/functional domains: A N-terminal domain spanning residues 1-191 that is responsible for apoE’s LDL receptor binding activity and a C-terminal domain (residues 216-199) that is responsible for lipoprotein-binding (1). The x-ray crystal structure of the lipid-free apoE N-terminal domain was solved by Wilson et al in 1991 which represented the only high-resolution structure of this protein. This structure showed an unusually elongated (65 Å) four-helix bundle (2) that was organized in such 2 a way that its hydrophobic faces were directed towards the protein interior, whereas the hydrophilic faces were oriented towards the solvent. The major receptor-binding region, residues 130-150, was located on the fourth helix. The amphipathic a-helices were connected by short loops, giving rise to a compact, globular structure. However, this structure only contained residues 23-165. Recent studies have shown that residues beyond residues 23-165 are also very important to the apoE LDL receptorbinding activity. For example, a mutation at position R172 reduces the receptor binding activity of apoE to only ~2% (3). In addition, an E3K mutant significantly increased the apoE receptor binding activity as well (4). While the x-ray crystal structure of the apoE N-terminal domain provided detailed structural information for most region of this domain, this structure does not provide an explanation of the above experimental results regarding the structural contribution to apoE’s LDL receptor binding activity by these residues that are located in the region which is not seen in the x-ray crystal structure.

Hoyt DW, SD Burton, MR Peterson, JD Myers, and G Chin, JR. 2004. "Expanding Your Laboratory by Accessing Collaboratory Resources." Analytical and Bioanalytical Chemistry 378(6):1408-1410. Abstract The Environmental Molecular Sciences Laboratory (EMSL) in Richland, Washington, is the home of a research facility setup by the United States Department of Energy (DOE). The facility is atypical because it houses over 100 cutting-edge research systems for the use of researchers all over the United States and the world. Access to the lab is requested through a peer-review proposal process and the scientists who use the facility are generally referred to as ‘users’. There are six main research facilities housed in EMSL, all of which host visiting researchers. Several of these facilities also participate in the EMSL Collaboratory, a remote access capability supported by EMSL operations funds. Of these, the High-Field Magnetic Resonance Facility (HFMRF) and Molecular Science Computing Facility (MSCF) have a significant number of their users performing remote work. The HFMRF in EMSL currently houses 12 NMR spectrometers that range in magnet field strength from 7.05T to 21.1T. Staff associated with the NMR facility offers scientific expertise in the areas of structural biology, solid-state materials/catalyst characterization, and magnetic resonance imaging (MRI) techniques. The way in which the HFMRF operates, with a high level of dedication to remote operation across the full suite of High-Field NMR spectrometers, has earned it the name “Virtual NMR Facility”. This review will focus on the operational aspects of remote research done in the High-Field Magnetic Resonance Facility and the computer tools that make remote experiments possible.