Jeff Abramson

The X-ray crystal structures of wild-type and EQ(I-286) mutant cytochrome c oxidases from Rhodobacter sphaeroides.

The structure of cytochrome c oxidase from Rhodobacter sphaeroides has been solved at 2.3/2.8A (anisotropic resolution). This high-resolution structure revealed atomic details of a bacterial terminal oxidase including water molecule positions and a potential oxygen pathway, which has not been reported in other oxidase structures. A comparative study of the wild-type and the EQ(I-286) mutant enzyme revealed structural rearrangements around E(I-286) that could be crucial for proton transfer in this enzyme. In the structure of the mutant enzyme, EQ(I-286), which cannot transfer protons during oxygen reduction, the side-chain of Q(I-286) does not have the hydrogen bond to the carbonyl oxygen of M(I-107) that is seen in the wild-type structure. Furthermore, the Q(I-286) mutant has a different arrangement of water molecules and residues in the vicinity of the Q side-chain. These differences between the structures could reflect conformational changes that take place upon deprotonation of E(I-286) during turnover of the wild-type enzyme, which could be part of the proton-pumping machinery of the enzyme.

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site.

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.

Lactose permease as a paradigm for membrane transport proteins (Review)

Our structural knowledge of the major facilitator superfamily (MFS) has dramatically increased in the past year with three structures of proteins from the MFS (oxalate/formate antiporter; lactose/proton symporter and the Pi/glycerol-3-phosphate antiporter). All three structures revealed 12 transmembrane helices forming two distinct domains and could imply that members of the MFS have preserved both secondary as well as tertiary structural elements during evolution. Lactose permease, a particularly well-studied member of the MFS, has been extensively explored by a number of molecular biological, biochemical and biophysical approaches. In this review, we take a closer look at the structure of LacY and incorporate a wealth of biochemical and biophysical data in order to propose a possible mechanism for lactose/proton symport. In addition, we make some brief comparisons between the structures of LacY and GlpT.

The Crystal Structure of the Primary Ca2+ Sensor of the Na+/Ca2+ Exchanger Reveals a Novel Ca2+ Binding Motif

The Na+/Ca2+ exchanger is a plasma membrane protein that regulates intracellular Ca2+ levels in cardiac myocytes. Transport activity is governed by Ca2+, and the primary Ca2+ sensor (CBD1) is located in a large cytoplasmic loop connecting two transmembrane helices. The binding of Ca2+ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca2+. Here, we present a crystal structure of CBD1 at 2.5Å resolution, which reveals a novel Ca2+ binding site consisting of four Ca2+ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca2+ binding cluster is indicative of a highly sensitive Ca2+ sensor and may represent a general platform for Ca2+ sensing.

The lactose permease of Escherichia coli: overall structure, the sugar‐binding site and the alternating access model for transport

Membrane transport proteins transduce free energy stored in electrochemical ion gradients into a concentration gradient and are a major class of membrane proteins, many of which play important roles in human health and disease. Recently, the X‐ray structure of the Escherichia coli lactose permease (LacY), an intensively studied member of a large group of related membrane transport proteins, was solved at 3.5 Å. LacY is composed of N‐ and C‐terminal domains, each with six transmembrane helices, symmetrically positioned within the molecule. The structure represents the inward‐facing conformation, as evidenced by a large internal hydrophilic cavity open to the cytoplasmic side. The structure with a bound lactose homolog reveals the sugar‐binding site in the cavity, and a mechanism for translocation across the membrane is proposed in which the sugar‐binding site has alternating accessibility to either side of the membrane.

Post-translational Modifications of Integral Membrane Proteins Resolved by Top-down Fourier Transform Mass Spectrometry with Collisionally Activated Dissociation

Integral membrane proteins remain a challenge to proteomics because they contain domains with physicochemical properties poorly suited to todays bottom-up protocols. These transmembrane regions may potentially contain post-translational modifications of functional significance, and thus development of protocols for improved coverage in these domains is important. One way to achieve this goal is by using top-down mass spectrometry whereby the intact protein is subjected to mass spectrometry and dissociation. Here we describe top-down high resolution Fourier transform mass spectrometry with collisionally activated dissociation to study post-translationally modified integral membrane proteins with polyhelix bundle and transmembrane porin motifs and molecular masses up to 35 kDa. On-line LC-MS analysis of the bacteriorhodopsin holoprotein yielded b- and y-ions that covered the full sequence of the protein and cleaved 79 of 247 peptide bonds (32%). The experiment proved that the mature sequence consists of residues 14–261, confirming N-terminal propeptide cleavage and conversion of N-terminal Gln-14 to pyrrolidone carboxylic acid (−17.02 Da) and C-terminal removal of Asp-262. Collisionally activated dissociation fragments localized the N6-(retinylidene) modification (266.20 Da) between residues 225–248 at Lys-229, the sole available amine in this stretch. Off-line nanospray of all eight subunits of the cytochrome b6f complex from the cyanobacterium Nostoc PCC 7120 defined various post-translational modifications, including covalently attached c-hemes (615.17 Da) on cytochromes f and b. Analysis of murine mitochondrial voltage-dependent anion channel established the amenability of the transmembrane β-barrel to top-down MS and localized a modification site of the inhibitor Ro 68-3400 at Cys-232. Where neutral loss of the modification is a factor, only product ions that carry the modification should be used to assign its position. Although bond cleavage in some transmembrane α-helical domains was efficient, other regions were refractory such that their primary structure could only be inferred from the coincidence of genomic translation with precursor and product ions that spanned them.

The second Ca2+-binding domain of the Na+–Ca2+ exchanger is essential for regulation: Crystal structures and mutational analysis

The Na+–Ca2+ exchanger plays a central role in cardiac contractility by maintaining Ca2+ homeostasis. Two Ca2+-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca2+ binding to these regulatory domains activates the transport of Ca2+ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca2+ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca2+-bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca2+ ions in primary and secondary Ca2+ sites. In the absence of Ca2+, Lys585 stabilizes the structure by coordinating two acidic residues (Asp552 and Glu648), one from each of the Ca2+-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca2+ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca2+ site completely remove Ca2+ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na+–Ca2+ exchanger.

Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1)

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca2+ release-activated Ca2+ (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca2+ sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca2+-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca2+ binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca2+-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.

Gated access to the pore of a P2X receptor: Structural implications for closed-open transitions

P2X receptors are ligand-gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor directly reveals that the ion-conducting pathway is formed by three transmembrane domain 2 (TM2) α-helices, each being provided by the three subunits of the trimer. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In this study, we quantified gated access to Cd2+ at substituted cysteines in TM2 of P2X2 receptors in the open and closed states. Our data for the closed state are consistent with the zebrafish P2X4.1 structure, with isoleucines and threonines (Ile-332 and Thr-336) positioned one helical turn apart lining the channel wall on approach to the gate. Our data for the open state reveal gated access to deeper parts of the pore (Thr-339, Val-343, Asp-349, and Leu-353), suggesting the closed channel gate is between Thr-336 and Thr-339. We also found unexpected interactions between native Cys-348 and D349C that result in tight Cd2+ binding deep within the intracellular vestibule in the open state. Interpreted with a P2X2 receptor structural model of the closed state, our data suggest that the channel gate opens near Thr-336/Thr-339 and is accompanied by movement of the pore-lining regions, which narrow toward the cytosolic end of TM2 in the open state. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP.

Structure-guided simulations illuminate the mechanism of ATP transport through VDAC1.

The voltage-dependent anion channel (VDAC) mediates the flow of metabolites and ions across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, whereas the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics simulations to construct a Markov state model of ATP permeation. These simulations indicate that ATP flows through VDAC through multiple pathways, in agreement with our structural data and experimentally determined physiological rates.