Michael D. Purdy

Outer Membrane Active Transport: Structure of the BtuB:TonB Complex

In Gram-negative bacteria, the import of essential micronutrients across the outer membrane requires a transporter, an electrochemical gradient of protons across the inner membrane, and an inner membrane protein complex (ExbB, ExbD, TonB) that couples the proton-motive force to the outer membrane transporter. The inner membrane protein TonB binds directly to a conserved region, called the Ton-box, of the transporter. We solved the structure of the cobalamin transporter BtuB in complex with the C-terminal domain of TonB. In contrast to its conformations in the absence of TonB, the Ton-box forms a β strand that is recruited to the existing β sheet of TonB, which is consistent with a mechanical pulling model of transport.

An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.

Crystal structure of an HIV assembly and maturation switch.

Virus assembly and maturation proceed through the programmed operation of molecular switches, which trigger both local and global structural rearrangements to produce infectious particles. HIV-1 contains an assembly and maturation switch that spans the C-terminal domain (CTD) of the capsid (CA) region and the first spacer peptide (SP1) of the precursor structural protein, Gag. The crystal structure of the CTD-SP1 Gag fragment is a goblet-shaped hexamer in which the cup comprises the CTD and an ensuing type II β-turn, and the stem comprises a 6-helix bundle. The β-turn is critical for immature virus assembly and the 6-helix bundle regulates proteolysis during maturation. This bipartite character explains why the SP1 spacer is a critical element of HIV-1 Gag but is not a universal property of retroviruses. Our results also indicate that HIV-1 maturation inhibitors suppress unfolding of the CA-SP1 junction and thereby delay access of the viral protease to its substrate. DOI: http://dx.doi.org/10.7554/eLife.17063.001

Use of a crystallization robot to set up sitting-drop vapor-diffusion crystallization and in situ crystallization screens

A commercial crystallization robot has been modified for use in setting up sitting-drop vapor-diffusion crystallization experiments, and for setting up protein crystallization screens in situ. The primary aim of this effort is the automated screening of crystallization of integral membrane proteins in detergent-containing solutions. However, the results of this work are of general utility to robotic liquid-handling systems. Sources of error that can prevent the accurate dispensing and mixing of solutions have been identified, and include local environmental, machine-specific and solution conditions. Solutions to each of these problems have been developed and implemented.

The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.

Design and characterization of a constitutively open KcsA

The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117–122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening.

Preliminary crystallographic analysis of the complex of the human GTPase Rhoa with the DH/PH tandem of PDZ-RhoGEF

PDZ-containing RhoGEF (PDZ-RhoGEF) is a multidomain protein composed of 1522 amino acids that belongs to the guanine nucleotide exchange factors family (GEF) active on Rho GTPases. It is highly specific for RhoA and is thought to transduce signals from Galpha(12/13)-coupled receptors to the RhoA-dependent regulatory cascades. The protein shows high sequence homology to LARG, p115-RhoGEF and Drosophila DRhoGEF2. The exchange reaction is catalyzed by a DH domain, which is directly downstream of a PH domain in all known Rho-specific GEFs. The DH/PH tandem of PDZ-RhoGEF and C-terminally truncated RhoA were overexpressed in Escherichia coli as TEV protease-cleavable fusion proteins containing GST and a hexahistidine tag at the N-termini, respectively. The nucleotide-free DH/PH-RhoA complex was purified by gel filtration and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 88.6, b = 119.0, c = 91.5 A, beta = 114.7 degrees.

Function and dynamics of macromolecular complexes explored by integrative structural and computational biology

Three vignettes exemplify the potential of combining EM and X-ray crystallographic data with molecular dynamics (MD) simulation to explore the architecture, dynamics and functional properties of multicomponent, macromolecular complexes. The first two describe how EM and X-ray crystallography were used to solve structures of the ribosome and the Arp2/3-actin complex, which enabled MD simulations that elucidated functional dynamics. The third describes how EM, X-ray crystallography, and microsecond MD simulations of a GPCR:G protein complex were used to explore transmembrane signaling by the β-adrenergic receptor. Recent technical advancements in EM, X-ray crystallography and computational simulation create unprecedented synergies for integrative structural biology to reveal new insights into heretofore intractable biological systems.

Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (HbαX)

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named Hb&agr;X) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, Hb&agr;X administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II–induced hypertensive states. Hb&agr;X acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O2 binding in red blood cells. In human vasculature, Hb&agr;X blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, Hb&agr;X modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature.

Recapitulation of metabolic defects in a model of propionic acidemia using patient-derived primary hepatocytes.

BACKGROUND Propionic acidemia (PA) is a disorder of intermediary metabolism with defects in the alpha or beta subunits of propionyl CoA carboxylase (PCCA and PCCB respectively) enzyme. We previously described a liver culture system that uses liver-derived hemodynamic blood flow and transport parameters to restore and maintain primary human hepatocyte biology and metabolism utilizing physiologically relevant milieu concentrations. METHODS In this study, primary hepatocytes isolated from the explanted liver of an 8-year-old PA patient were cultured in the liver system for 10 days and evaluated for retention of differentiated polarized morphology. The expression of PCCA and PCCB was assessed at a gene and protein level relative to healthy donor controls. Ammonia and urea levels were measured in the presence and absence of amino acid supplements to assess the metabolic consequences of branched-chain amino acid metabolism in this disease. RESULTS Primary hepatocytes from the PA patient maintained a differentiated polarized morphology (peripheral actin staining) over 10 days of culture in the system. We noted lower levels of PCCA and PCCB relative to normal healthy controls at the mRNA and protein level. Supplementation of branched-chain amino acids, isoleucine (5mM) and valine (5mM) in the medium, resulted in increased ammonia and decreased urea in the PA patient hepatocyte system, but no such response was seen in healthy hepatocytes or patient-derived fibroblasts. CONCLUSIONS We demonstrate for the first time the successful culture of PA patient-derived primary hepatocytes in a differentiated state, that stably retain the PCCA and PCCB enzyme defects at a gene and protein level. Phenotypic response of the system to an increased load of branched-chain amino acids, not possible with fibroblasts, underscores the utility of this system in the better understanding of the molecular pathophysiology of PA and examining the effectiveness of potential therapeutic agents in the most relevant tissue.