Kevin L. Weiss

Low resolution structure and dynamics of a Colicin-Receptor complex determined by neutron scattering

Background: In order to kill E. coli, colicins need to cross the bacterial outer membrane. Results: Neutron scattering data show colicin N at the protein-lipid interface of its receptor OmpF. Conclusion: Colicins can unfold and penetrate membranes via the outside wall of their receptors. Significance: The protein-lipid interface may be the route that colicins take into the cell. Proteins that translocate across cell membranes need to overcome a significant hydrophobic barrier. This is usually accomplished via specialized protein complexes, which provide a polar transmembrane pore. Exceptions to this include bacterial toxins, which insert into and cross the lipid bilayer itself. We are studying the mechanism by which large antibacterial proteins enter Escherichia coli via specific outer membrane proteins. Here we describe the use of neutron scattering to investigate the interaction of colicin N with its outer membrane receptor protein OmpF. The positions of lipids, colicin N, and OmpF were separately resolved within complex structures by the use of selective deuteration. Neutron reflectivity showed, in real time, that OmpF mediates the insertion of colicin N into lipid monolayers. This data were complemented by Brewster Angle Microscopy images, which showed a lateral association of OmpF in the presence of colicin N. Small angle neutron scattering experiments then defined the three-dimensional structure of the colicin N-OmpF complex. This revealed that colicin N unfolds and binds to the OmpF-lipid interface. The implications of this unfolding step for colicin translocation across membranes are discussed.

The Bio-SANS instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory

Small-angle neutron scattering (SANS) is a powerful tool for characterizing complex disordered materials, including biological materials. The Bio-SANS instrument of the High Flux Isotope Reactor of Oak Ridge National Laboratory (ORNL) is a high-flux low-background SANS instrument that is, uniquely among SANS instruments, dedicated to serving the needs of the structural biology and biomaterials communities as an open-access user facility. Here, the technical specifications and performance of the Bio-SANS are presented. Sample environments developed to address the needs of the user program of the instrument are also presented. Further, the isotopic labeling and sample preparation capabilities available in the Bio-Deuteration Laboratory for users of the Bio-SANS and other neutron scattering instruments at ORNL are described. Finally, a brief survey of research performed using the Bio-SANS is presented, which demonstrates the breadth of the research that the instruments user community engages in.

The active site protonation states of perdeuterated Toho‐1 β‐lactamase determined by neutron diffraction support a role for Glu166 as the general base in acylation

Room temperature neutron diffraction data of the fully perdeuterated Toho‐1 R274N/R276N double mutant β‐lactamase in the apo form were used to visualize deuterium atoms within the active site of the enzyme. This perdeuterated neutron structure of the Toho‐1 R274N/R276N reveals the clearest picture yet of the ground‐state active site protonation states and the complete hydrogen‐bonding network in a β‐lactamase enzyme. The ground‐state active site protonation states detailed in this neutron diffraction study are consistent with previous high‐resolution X‐ray studies that support the role of Glu166 as the general base during the acylation reaction in the class A β‐lactamase reaction pathway.

Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography.

The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 A resolution neutron diffraction studies of fully perdeuterated and selectively CH(3)-protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 A resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the sigma level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 A resolution RT neutron data for perdeuterated rubredoxin are approximately 8 times more likely overall to provide high-confidence positions for D atoms than 1.1 A resolution X-ray data at 100 K or RT. At or above the 1.0sigma level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 A resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0sigma level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.

Neutron and X-ray crystal structures of a perdeuterated enzyme inhibitor complex reveal the catalytic proton network of the Toho-1 β-lactamase for the acylation reaction

Background: Antibiotic resistance from extended-spectrum β-lactamases (ESBLs) makes infections more dangerous and difficult to treat. Results: Neutron and x-ray crystal structures were determined for an ESBL in complex with an acylation transition state analog. Conclusion: Glu-166 is implicated as the general base in the acylation reaction. Significance: Understanding the catalytic mechanism of β-lactamases will lead to improved antibiotics and β-lactamase inhibitors. The mechanism by which class A β-lactamases hydrolyze β-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the β-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 β-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with 11B, as 10B is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction.

Rapid visualization of hydrogen positions in protein neutron crystallographic structures

Neutron crystallography is a powerful technique for experimental visualization of the positions of light atoms, including hydrogen and its isotope deuterium. In recent years, structural biologists have shown increasing interest in the technique as it uniquely complements X-ray crystallographic data by revealing the positions of D atoms in macromolecules. With this regained interest, access to macromolecular neutron crystallography beamlines is becoming a limiting step. In this report, it is shown that a rapid data-collection strategy can be a valuable alternative to longer data-collection times in appropriate cases. Comparison of perdeuterated rubredoxin structures refined against neutron data sets collected over hours and up to 5 d shows that rapid neutron data collection in just 14 h is sufficient to provide the positions of 269 D atoms without ambiguity.

X-ray crystallographic studies of family 11 xylanase Michaelis and product complexes: implications for the catalytic mechanism

Xylanases catalyze the hydrolysis of plant hemicellulose xylan into oligosaccharides by cleaving the main-chain glycosidic linkages connecting xylose subunits. To study ligand binding and to understand how the pH constrains the activity of the enzyme, variants of the Trichoderma reesei xylanase were designed to either abolish its activity (E177Q) or to change its pH optimum (N44H). An E177Q-xylohexaose complex structure was obtained at 1.15 Å resolution which represents a pseudo-Michaelis complex and confirmed the conformational movement of the thumb region owing to ligand binding. Co-crystallization of N44H with xylohexaose resulted in a hydrolyzed xylotriose bound in the active site. Co-crystallization of the wild-type enzyme with xylopentaose trapped an aglycone xylotriose and a transglycosylated glycone product. Replacing amino acids near Glu177 decreased the xylanase activity but increased the relative activity at alkaline pH. The substrate distortion in the E177Q-xylohexaose structure expands the possible conformational itinerary of this xylose ring during the enzyme-catalyzed xylan-hydrolysis reaction.

Neutron crystal structure of RAS GTPase puts in question the protonation state of the GTP γ-phosphate

Background: The GTP nucleotide is thought to be fully deprotonated when bound to RAS. Results: The neutron crystal structure of RAS bound to the GTP analogue GppNHp shows a protonated γ-phosphate. Conclusion: The active site of RAS significantly increases the pKa of the nucleotide. Significance: This work provides insight to the GTP hydrolysis mechanism, with implications to the superfamily of small GTPases. RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.

Cryogenic neutron protein crystallography: routine methods and potential benefits

The use of cryocooling in neutron diffraction has been hampered by several technical challenges, such as the need for specialized equipment and techniques. This article reports the recent development and deployment of equipment and strategies that allow routine neutron data collection on cryocooled crystals using off-the-shelf components. This system has several advantages compared to a closed displex cooling system, such as fast cooling coupled with easier crystal mounting and centering. The ability to routinely collect cryogenic neutron data for analysis will significantly broaden the range of scientific questions that can be examined by neutron protein crystallography. Cryogenic neutron data collection for macromolecules has recently become available at the new Biological Diffractometer BIODIFF at the FRM II and the Macromolecular Diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge National Laboratory. To evaluate the benefits of a cryocooled neutron structure, a full neutron data set was collected on the BIODIFF instrument on a Toho-1 β-lactamase structure at 100 K.

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

The catalytic mechanism of class A β-lactamases is often debated due in part to the large number of amino acids that interact with bound β-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type β-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 β-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.