Fang Tian

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.

In Situ Structural Characterization of a Recombinant Protein in Native Escherichia coli Membranes with Solid-State Magic-Angle-Spinning NMR

The feasibility of using solid-state magic-angle-spinning NMR spectroscopy for in situ structural characterization of the LR11 (sorLA) transmembrane domain (TM) in native Escherichia coli membranes is presented. LR11 interacts with the human amyloid precursor protein (APP), a central player in the pathology of Alzheimers disease. The background signals from E. coli lipids and membrane proteins had only minor effects on the LR11 TM resonances. Approximately 50% of the LR11 TM residues were assigned by using (13)C PARIS data. These assignments allowed comparisons of the secondary structure of the LR11 TM in native membrane environments and commonly used membrane mimics (e.g., micelles). In situ spectroscopy bypasses several obstacles in the preparation of membrane proteins for structural analysis and offers the opportunity to investigate how membrane heterogeneity, bilayer asymmetry, chemical gradients, and macromolecular crowding affect the protein structure.

Presentation of Membrane-Anchored Glycosphingolipids Determined from Molecular Dynamics Simulations and NMR Paramagnetic Relaxation Rate Enhancement

Challenges for structural characterization of membrane-bound glycosphingolipids include their high internal dynamic motions and their physical proximity to membrane surfaces. Here we demonstrate that NMR paramagnetic relaxation enhancement can be used, alongside independent molecular dynamics simulations and an outer-sphere relaxation model, to quantitatively characterize the presentation (insertion depth and orientation relative to a membrane surface) of ganglioside GM1 in biologically relevant membrane environments. Longitudinal and transverse paramagnetic relaxation enhancement effects were measured for GM1, anchored to phospholipid bicelles, using both water-soluble and membrane-anchored paramagnetic probes, respectively. A method was developed to rapidly calculate paramagnetic relaxation enhancement effects from thousands of structures taken from a simulation of GM1 in a phospholipid bilayer. The combined computational and experimental approach yielded experimentally verified atomic-resolution 3D models of a highly plastic membrane-bound biomolecule.

Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS)

Residual dipolar couplings are being increasingly used as structural constraints for NMR studies of biomolecules. A problem arises when dipolar coupling contributions are larger than scalar contributions for a given spin pair, as is commonly observed in solid state NMR studies, in that signs of dipolar couplings cannot easily be determined. Here the sign ambiguities of dipolar couplings in field-oriented bicelles are resolved by variable angle sample spinning (VASS) techniques. The director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS is studied by 31P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7°, and the director (or bicelle normal) of the disks is mainly distributed in a plane perpendicular to the rotation axis. Since the dipolar couplings depend on how the bicelles are oriented with respect to the magnetic field, it is shown that the dipolar interaction can be scaled to the same order as the J-coupling by moving the spinning axis from 0° toward 54.7°. Thus the relative sign of dipolar and scalar couplings can be determined.

Structural basis for the geometry-driven localization of a small protein.

Significance Despite extensive studies of protein trafficking across length scales of many microns, how proteins correctly localize within the smaller length scales of bacterial cells is still poorly understood. Recently, we proposed that slight membrane curvature, defined by the surface geometry of a bacterium, can drive the localization of certain shape-sensing proteins. Here, we developed an assay to quantify membrane curvature recognition by the small bacterial protein SpoVM and used NMR to determine the structural basis of curvature recognition. NMR and molecular dynamics simulations suggested a model wherein unusually deep membrane insertion allows the protein to sense subtle acyl chain packing differences between differently curved membranes, a distinct curvature-sensing mechanism from those used by proteins that sense high membrane curvature. In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVMP9A) to reveal that SpoVM’s atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM’s membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.

REDCRAFT: a tool for simultaneous characterization of protein backbone structure and motion from RDC data.

REDCRAFT, a new open source software tool that accommodates the analysis of RDC data for simultaneous structure and dynamics characterization of proteins is presented in this article. Simultaneous consideration of structure and motion is believed to be necessary for accurate representation of the solution-state of a protein. REDCRAFT is designed to primarily utilize RDC data from multiple alignment media in two stages. During Stage-I, a list of possible torsion angles joining any two neighboring peptide planes is ranked based on their fitness to experimental constraints; in Stage-II, a dipeptide fragment is extended by addition of one peptide plane at a time. The algorithm adopted by REDCRAFT is very efficient and can produce a structure for an 80 residue protein within two hours on a typical desktop computer. REDCRAFT exhibits robustness with respect to noise and missing data. REDCRAFT describes the overall alignment of the molecule in the form of an order tensor matrix and is capable of identifying peptide fragments with internal dynamics. Identification of the location of internal motion will permit a more accurate structural representation. Experimental data from two proteins as well as simulated data are presented to illustrate the capabilities of REDCRAFT in both structure determination and identification of the dynamical regions.

Direct measurement of 1H-1H dipolar couplings in proteins: a complement to traditional NOE measurements.

An intensity-based constant-time COSY (CT-COSY) method is described for measuring 1H-1H residual dipolar couplings of proteins in weakly aligned media. For small proteins, the overall sensitivity of this experiment is comparable to the NOESY experiment. In cases where the 1H-1H distances are defined by secondary structure, such as 1Hα-1HN and 1HN-1HN sequential distances in α-helices and β-sheets, these measurements provide useful orientational constraints for protein structure determination. This experiment can also be used to provide distance information similar to that obtained from NOE connectivities once the angular dependence is removed. Because the measurements are direct and non-coherent processes, such as spin diffusion, do not enter, the measurements can be more reliable. The 1/r3 distance dependence of directly observed dipolar couplings, as compared with the 1/r6 distance dependence of NOEs, also can provide longer range distance information at favorable angles. A simple 3D, 15N resolved version of the pulse sequence extends the method to provide the improved resolution required for application to larger biomolecules.

The occludin and ZO-1 complex, defined by small angle X-ray scattering and NMR, has implications for modulating tight junction permeability

Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468–475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin∶ZO-1 complex.

Structure and dynamics from solid-state NMR spectroscopy

The authors wish to thank W Thorner for his skill in preparing the colour figure for this manuscript. TAC gratefully acknowledges support of the National Science Foundation (DMB931711) and the National High Magnetic Field Laboratory at Florida State University.

Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro

ABSTRACT Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming.