Jan K. Rainey

Structural and functional analysis of the Na+/H+ exchanger.

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.

Structural and functional characterization of transmembrane segment VII of the Na(+)/H(+) exchanger isoform 1

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H+ for one extracellular Na+. It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251–273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na+/H+ exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly α-helical, with a break in the helix at the functionally critical residues Gly261–Glu262. The relative positions and orientations of the N- and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition.

Fibrous Long Spacing Collagen Ultrastructure Elucidated by Atomic Force Microscopy

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.

Structural insight into G-protein coupled receptor binding by apelin.

Apelin peptides are the cognate ligands for the G-protein coupled receptor APJ, with functions in the cardiovascular and central nervous systems, in glucose metabolism and as a human immunodeficiency virus (HIV-1) coreceptor. Apelin is found in 13-36 residue forms in vivo. The structures of five isoforms of apelin at physiological versus low (5-6 degrees C) temperature are compared here using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, demonstrating increased structure at low temperature. Far-ultraviolet (UV) CD spectra are predominantly random coil for apelin isoforms, but are convoluted by unusual bands from the C-terminal phenylalanine side chain. These bands, assigned using F13A-apelin-13, are accentuated at 5 degrees C and imply conformational restriction. At 35 degrees C, the R6-L9 region of apelin-17 is well structured, consistent with previous mutagenesis results showing necessity of this segment for apelin-APJ binding and activation. At 5 degrees C, R6-L9 retains its structuring while the functionally critical C-terminal G13-F17 region also becomes highly structured. Type IV beta-turns and some polyproline-II structure alongside F17 side chain motional reduction correlate well with CD spectral properties. Cis-trans peptide bond isomerization at P14 and P16 produces two sequentially assignable conformers (both trans:both cis approximately 4:1) alongside less populated conformers. Chemical shift assignment of apelin-12, -13 and pyroglutamate-apelin-13 implies highly similar structuring and the same isomerization at the C-terminus. Based on the apelin-17 structure, a two-step binding and activation mechanism is hypothesized.

A statistically derived parameterization for the collagen triple-helix.

The triple‐helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo‐ or hetero‐tripeptide with a repeating primary sequence of (Gly‐X‐Y)n, displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple‐helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most collagen molecules are primarily triple‐helical; however, no high‐resolution structure of any entire protein is yet available. Given the drastic morphological differences in self‐assembled collagen structures with subtle changes in assembly conditions, a detailed knowledge of the relative locations of charged and sterically bulky residues in collagen is desirable. Its repetitive primary sequence and highly conserved secondary structure make collagen, and the triple‐helix in general, an ideal candidate for a general parameterization for prediction of residue locations and for the use of a helical wheel in the prediction of residue orientation. Herein, a statistical analysis of the currently available high‐resolution X‐ray crystal structures of model triple‐helical peptides is performed to produce an experimentally based parameter set for predicting peptide backbone and Cβ atom locations for the triple‐helix. Unlike existing homology models, this allows easy prediction of an entire triple‐helix structure based on all existing high‐resolution triple‐helix structures, rather than only on a single structure or on idealized parameters. Furthermore, regional differences based on the helical propensity of residues may be readily incorporated. The parameter set is validated in terms of the predicted bond lengths, backbone dihedral angles, and interchain hydrogen bonding.

Improved Helix and Kink Characterization in Membrane Proteins Allows Evaluation of Kink Sequence Predictors

Although the α-helical secondary structure of proteins is well-defined, the exact causes and structures of helical kinks are not. This is especially important for transmembrane (TM) helices of integral membrane proteins, many of which contain kinks providing functional diversity despite predominantly helical structure. We have developed a Monte Carlo method based algorithm, MC-HELAN, to determine helical axes alongside positions and angles of helical kinks. Analysis of all nonredundant high-resolution α-helical membrane protein structures (842 TM helices from 205 polypeptide chains) revealed kinks in 64% of TM helices, demonstrating that a significantly greater proportion of TM helices are kinked than those indicated by previous analyses. The residue proline is over-represented by a factor >5 if it is two or three residues C-terminal to a bend. Prolines also cause kinks with larger kink angles than other residues. However, only 33% of TM kinks are in proximity to a proline. Machine learning techniques were used to test for sequence-based predictors of kinks. Although kinks are somewhat predicted by sequence, kink formation appears to be driven predominantly by other factors. This study provides an improved view of the prevalence and architecture of kinks in helical membrane proteins and highlights the fundamental inaccuracy of the typical topological depiction of helical membrane proteins as series of ideal helices.

The apelin receptor: Physiology, pathology, cell signalling, and ligand modulation of a peptide-activated class A GPCR

The apelin receptor (AR or APJ) is a class A (rhodopsin-like) G-protein-coupled receptor with wide distribution throughout the human body. Activation of the AR by its cognate peptide ligand, apelin, induces diverse physiological effects including vasoconstriction and dilation, strengthening of heart muscle contractility, angiogenesis, and regulation of energy metabolism and fluid homeostasis. Recently, another endogenous peptidic activator of the AR, Toddler/ELABELA, was identified as having a crucial role in zebrafish (Danio rerio) embryonic development. The AR is also implicated in pathologies including cardiovascular disease, diabetes, obesity, and cancer, making it a promising therapeutic target. Despite its established importance, the precise roles of AR signalling remain poorly understood. Moreover, little is known about the mechanisms of peptide-AR activation. Additional complexity arises from modulation of the AR by 2 endogenous peptide ligands, both with multiple bioactive isoforms of variable length and distribution. The various apelin and Toddler/ELABELA isoforms may also produce distinct cellular effects. Further complexity arises through formation of functionally distinct heterodimers between the AR and other G-protein-coupled receptors. This minireview outlines key (patho)physiological actions of the AR, addresses what is known about signal transduction downstream of AR activation, and concludes by discussing unique properties of the endogenous peptidic ligands of the AR.

Recombinant Minimalist Spider Wrapping Silk Proteins Capable of Native-Like Fiber Formation

Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W1 to W4. We observed that purified W2, W3 and W4 proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W4 were ∼3.4 µm in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm−3. The smaller W2 protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W1 protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly α-helical in solution to increasingly β-sheet in fibers.

Headgroup-dependent membrane catalysis of apelin-receptor interactions is likely.

Apelin is the peptidic ligand for the G-protein-coupled receptor APJ. The apelin−APJ system is important in cardiovascular regulation, fluid homeostasis, and angiogenesis, among other roles. In this study, we investigate interactions between apelin and membrane-mimetic micelles of the detergents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG). Far-ultraviolet circular dichroism spectropolarimetry and diffusion-ordered spectroscopy indicate that apelin peptides bind to micelles of the anionic detergents SDS and LPPG much more favorably than to zwitterionic DPC micelles. Nuclear magnetic resonance spectroscopy allowed full characterization of the interactions of apelin-17 with SDS micelles. Titration with paramagnetic agents and structural determination of apelin-17 in SDS indicate that R6−K12 is highly structured, with R6−L9 directly interacting with headgroups of the micelle. Type I β-turns are initiated between R6 and L9, and a well-defined type IV β-turn is initiated at S10. Furthermore, binding of apelin-17 to SDS micelles causes structuring of M15-F17, with no evidence for direct binding of this region to the micelles. These results are placed into the context of the membrane catalysis hypothesis for peptide−receptor binding, and a hypothetical mechanism of APJ binding and activation by apelin is advanced.

Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.