Brent W. Segelke

The CD1 family of lipid antigen-presenting molecules

Abstract The paradigm that T cells recognize peptide antigens presented by major histocompatibility complex class I and class II molecules has been a guiding principle in the development of immunology. Here, Steven Porcelli and colleagues review studies that extend this paradigm by showing that CD1 proteins are a separate lineage of antigen-presenting molecules with unusually hydrophobic ligand-binding grooves that present nonpeptide lipid and glycolipid antigens to T cells.

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Fixed-target protein serial microcrystallography with an x-ray free electron laser

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Interaction of the N-terminal domain of apolipoprotein E4 with heparin.

Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimers disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimers disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->[4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->](3)4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.

Femtosecond X-ray diffraction from two-dimensional protein crystals

Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented.

The high-speed Hydra-Plus-One system for automated high-throughput protein crystallography

An automated high-throughput dispenser has been developed for the setup of protein crystallization trials by vapor diffusion or Microbatch methods. The Hydra-Plus-One is composed of a Hydra-PP system equipped with a motorized XYZ-platform, 96 precision glass syringes and a single-channel microsolenoid dispenser, which transfers 100 nl-50 micro l of protein solution with an accuracy of > 90% at a speed of 60s per 96 wells. Up to 300 micro l of premixed cocktails can be aspirated with the 96-syringe-assembly and dispensed into reservoir and droplet wells within 60s. The Hydra-Plus-One combines high precision, reliability and speed in a cost-effective high-throughput system ideally suited for protein crystallization

The TB structural genomics consortium crystallization facility: towards automation from protein to electron density.

The crystallization facility of the TB (Tuberculosis) structural genomics consortium, one of nine NIH sponsored p50 structural genomic centres, provides TB consortium members with automated crystallization, data collection and basic molecular replacement (MR) structure solution up to bias minimized electron density maps. Crystallization setup of up to ten proteins per day follows the CRYSTOOL combinatorial screen protocol using a modular and affordable robotic design with an open architecture. Components include screen preparation, plate setup, automated image acquisition and analysis, and optimisation design. A new 96 well crystallization plate has been designed for optimal robotic handling while maintaining ease of manual crystal harvesting. Robotic crystal mounting, screening, and data collection are conducted in-house and at the Advanced Light Source (ALS) in Berkeley. A simple automated protocol based on MR and homology based structure prediction automatically solves modestly difficult problems. Multiple search models are evaluated in parallel MR and the best multi-segment rigid body refined MR solution is subjected to simulated annealing torsion angle molecular dynamics using CNS, bringing even marginal MR solutions within the convergence radius of the subsequent highly effective bias removal and map reconstruction protocol, Shake&wARP, used to generate electron density for initial rebuilding. Real space correlation plots allow rapid assessment of local structure quality. Modular design of robotics and automated scripts using publicly available programs for structure solution allow for efficient high throughput crystallography - at a reasonable cost.

High throughput crystallography of TB drug targets.

Tuberculosis (TB) infects one-third of the world population. Despite 50 years of available drug treatments, TB continues to increase at a significant rate. The failure to control TB stems in part from the expense of delivering treatment to infected individuals and from complex treatment regimens. Incomplete treatment has fueled the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Reducing non-compliance by reducing the duration of chemotherapy will have a great impact on TB control. The development of new drugs that either kill persisting organisms, inhibit bacilli from entering the persistent phase, or convert the persistent bacilli into actively growing cells susceptible to our current drugs will have a positive effect. We are taking a multidisciplinary approach that will identify and characterize new drug targets that are essential for persistent Mtb. Targets are exposed to a battery of analyses including microarray experiments, bioinformatics, and genetic techniques to prioritize potential drug targets from Mtb for structural analysis. Our core structural genomics pipeline works with the individual laboratories to produce diffraction quality crystals of targeted proteins, and structural analysis will be completed by the individual laboratories. We also have capabilities for functional analysis and the virtual ligand screening to identify novel inhibitors for target validation. Our overarching goals are to increase the knowledge of Mtb pathogenesis using the TB research community to drive structural genomics, particularly related to persistence, develop a central repository for TB research reagents, and discover chemical inhibitors of drug targets for future development of lead compounds.

Questions about the structure of the botulinum neurotoxin B light chain in complex with a target peptide

Hanson and Stevens1 recently reported the cocrystal structure of the botulinum neurotoxin B light chain in complex with a 38-residue target peptide of synaptobrevin II. Based on a careful examination of the published data, we have serious concerns about the strength of experimental evidence supporting the presence of the target peptide, and consequently the validity of inferences about substrate binding made in the report.

Concanavalin A in a dimeric crystal form: revisiting structural accuracy and molecular flexibility

A structure of native concanavalin A (ConA), a hardy perennial of structural biology, has been determined in a dimeric crystal form at a resolution of 1.56 A (space group C222(1); unit-cell parameters a = 118.70, b = 101.38, c = 111.97 A; two molecules in the asymmetric unit). The structure has been refined to an R(free) of 0.206 (R = 0.178) after iterative model building and phase-bias removal using Shake&wARP. Correspondence between calculated water-tyrosine interactions and experimentally observed structures near the saccharide-binding site suggests that the observed interactions between Tyr12 and water in various crystal forms are to be expected and are not unique to the presence of an active site. The present structure differs from previously reported atomic resolution structures of ConA in several regions and extends insight into the conformational flexibility of this molecule. Furthermore, this third, low-temperature, structure of ConA in a different crystal form, independently refined using powerful model-bias removal techniques, affords the opportunity to revisit assessment of accuracy and precision in high- or atomic resolution protein structures. It is illustrated that several precise structures of the same molecule can differ substantially in local detail and users of crystallographic models are reminded to consider the potential impact when interpreting structures. Suggestions on how to effectively represent ensembles of crystallographic models of a given molecule are provided.