Wade D. Van Horn

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.

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.

Reverse Micelle Encapsulation as a Model for Intracellular Crowding

Reverse micelles are discrete nanoscale particles composed of a water core surrounded by surfactant. The amount of water within the core of reverse micelles can be easily manipulated to directly affect the size of the reverse micelle particle. The water loading capacity of reverse micelles varies with temperature, and water can be shed if reverse micelles are exposed to low temperatures. The use of water shedding from the reverse micelle provides precise and comprehensive control over the amount of water available to solvate host molecules. Proteins encapsulated within reverse micelles can be studied to determine the effects of confinement and excluded volume. The data presented here provide an important bridge between commonly employed dilute in vitro studies and studies of the effects of a crowded environment, as found in vivo. Ubiquitin was encapsulated within bis(2-ethylhexyl) sodium sulfosuccinate AOT reverse micelles under various degrees of confinement and was compared with an analogously reconstituted sample of ubiquitin in the commonly used molecular crowding agent bovine serum albumin. The effects of encapsulation were monitored using chemical shift perturbation analysis of the amide (1)H and (15)N resonances. The results also reconcile alternative interpretations of protein cold denaturation within reverse micelles.

Prokaryotic diacylglycerol kinase and undecaprenol kinase.

Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins-including water soluble kinases-and exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a by-product of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in gram-positive bacteria, where its importance is evident because UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic DAGK family, which is based on over 40 years of studies.

The structural diversity of artificial genetic polymers

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson–Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.

Bicelles at Low Concentrations

Bilayered detergent-lipid assemblies known as bicelles have been widely used as model membranes in structural biological studies and are being explored for wider applications, including pharmaceutical use. Most studies to date have involved the use of concentrated bicelle mixtures, such that little is known about the capacity of bicellar mixtures to be diluted without unwanted transitions to nonisotropic phases. Here, different detergent/lipid mixtures have been explored, leading to the identification of two different families of bicelles for which it is possible to lower the total amphiphile (detergent + lipid) concentration to <1% (w/v) while retaining isotropic assemblies. These include a novel family of bicelles based on mixtures of 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and the lipid dimyristoylphosphatidylcholine (DMPC). Bicelles formed by these mixtures can be diluted to <0.5% and also have attractive biochemical properties. However, a caveat of our results is that the diffusion coefficients measured for the lipid component of the different bicelles tested were seen to be dependent on sample history, even though all samples were optically transparent. This suggests that the phase behavior of bicelles at low lipid-to-detergent ratios may be more complex than previously appreciated.

The Quiet Renaissance of Protein Nuclear Magnetic Resonance

From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 1980s.

Understanding Thermosensitive Transient Receptor Potential Channels as Versatile Polymodal Cellular Sensors

Transient receptor potential (TRP) ion channels are eukaryotic polymodal sensors that function as molecular cellular signal integrators. TRP family members sense and are modulated by a wide array of inputs, including temperature, pressure, pH, voltage, chemicals, lipids, and other proteins. These inputs induce signal transduction events mediated by nonselective cation passage through TRP channels. In this review, we focus on the thermosensitive TRP channels and highlight the emerging view that these channels play a variety of significant roles in physiology and pathophysiology in addition to sensory biology. We attempt to use this viewpoint as a framework to understand the complexity and controversy of TRP channel modulation and ultimately suggest that the complex functional behavior arises inherently because this class of protein is exquisitely sensitive to many diverse and distinct signal inputs. To illustrate this idea, we primarily focus on TRP channel thermosensing. We also offer a structural, biochemical, biophysical, and computational perspective that may help to bring more coherence and consensus in understanding the function of this important class of proteins.

Working model for the structural basis for KCNE1 modulation of the KCNQ1 potassium channel

The voltage-gated potassium channel KCNQ1 (Kv7.1) is modulated by KCNE1 (minK) to generate the I(Ks) current crucial to heartbeat. Defects in either protein result in serious cardiac arrhythmias. Recently developed structural models of the open and closed state KCNQ1/KCNE1 complexes offer a compelling explanation for how KCNE1 slows channel opening and provides a platform from which to refine and test hypotheses for other aspects of KCNE1 modulation. These working models were developed using an integrative approach based on results from nuclear magnetic resonance spectroscopy, electrophysiology, biochemistry, and computational methods-an approach that can be applied iteratively for model testing and revision. We present a critical review of these structural models, illustrating the strengths and challenges of the integrative approach.

Functional delivery of a membrane protein into oocyte membranes using bicelles

Voltage-gated potassium channel modulatory membrane protein KCNE3 was overexpressed and purified into both micelles and bicelles. Remarkably, microinjection of KCNE3 in bicelles into Xenopus oocytes resulted in functional co-assembly with the human KCNQ1 channel expressed therein. Microinjection of LMPC micelles containing KCNE3 did not result in channel modulation, indicating that bicelles sometimes succeed at delivering a membrane protein into a cellular membrane when classical micelles fail. Backbone NMR resonance assignments were completed for KCNE3 in both bicelles and LMPC, indicating that the secondary structure distribution in KCNE3s N-terminus and transmembrane domains exhibits only modest differences from that of KCNE1, even though these KCNE family members have very different effects on KCNQ1 channel function.