Structure of fully protonated proteins by proton-detected magic-angle spinning NMR.

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of (1)H-(1)H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.

Dynamic Nuclear Polarization-Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic-Angle Spinning.

Dynamic nuclear polarization (DNP) is a powerful way to overcome the sensitivity limitation of magic-angle-spinning (MAS) NMR experiments. However, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals. High-quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast MAS (40 kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar-based experiments. This enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long-range contacts, which are not observed at room temperature, opening new possibilities for structure determination.

NMR Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 µg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning.

Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

Out-and-back 13C-13C scalar transfers in protein resonance assignment by proton-detected solid-state NMR under ultra-fast MAS.

We present here (1)H-detected triple-resonance H/N/C experiments that incorporate CO-CA and CA-CB out-and-back scalar-transfer blocks optimized for robust resonance assignment in biosolids under ultra-fast magic-angle spinning (MAS). The first experiment, (H)(CO)CA(CO)NH, yields (1)H-detected inter-residue correlations, in which we record the chemical shifts of the CA spins in the first indirect dimension while during the scalar-transfer delays the coherences are present only on the longer-lived CO spins. The second experiment, (H)(CA)CB(CA)NH, correlates the side-chain CB chemical shifts with the NH of the same residue. These high sensitivity experiments are demonstrated on both fully-protonated and 100%-H(N) back-protonated perdeuterated microcrystalline samples of Acinetobacter phage 205 (AP205) capsids at 60 kHz MAS.

Crystal structure of human gamma-butyrobetaine hydroxylase.

Gamma-butyrobetaine hydroxylase (GBBH) is a 2-ketoglutarate-dependent dioxygenase that catalyzes the biosynthesis of l-carnitine by hydroxylation of gamma-butyrobetaine (GBB). l-carnitine is required for the transport of long-chain fatty acids into mitochondria for generating metabolic energy. The only known synthetic inhibitor of GBBH is mildronate (3-(2,2,2-trimethylhydrazinium) propionate dihydrate), which is a non-hydroxylatable analog of GBB. To aid in the discovery of novel GBBH inhibitors by rational drug design, we have solved the three-dimensional structure of recombinant human GBBH at 2.0A resolution. The GBBH monomer consists of a catalytic double-stranded beta-helix (DBSH) domain, which is found in all 2KG oxygenases, and a smaller N-terminal domain. Extensive interactions between two monomers confirm earlier observations that GBBH is dimeric in its biological state. Although many 2KG oxygenases are multimeric, the dimerization interface of GBBH is very different from that of related enzymes. The N-terminal domain of GBBH has a similar fold to the DUF971 superfamily, which consists of several short bacterial proteins with unknown function. The N-terminal domain has a bound Zn ion, which is coordinated by three cysteines and one histidine. Although several other 2KG oxygenases with known structures have more than one domain, none of them resemble the N-terminal domain of GBBH. The N-terminal domain may facilitate dimer formation, but its precise biological role remains to be discovered. The active site of the catalytic domain of GBBH is similar to that of other 2KG oxygenases, and Fe(II)-binding residues form a conserved His-X-Asp-X(n)-His triad, which is found in all related enzymes.

Eliminating Factor H-Binding Activity of Borrelia burgdorferi CspZ Combined with Virus-Like Particle Conjugation Enhances Its Efficacy as a Lyme Disease Vaccine.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the most common tick-borne disease in the US and Europe. No potent human vaccine is currently available. The innate immune complement system is vital to host defense against pathogens, as complement activation on the surface of spirochetes results in bacterial killing. Complement system is inhibited by the complement regulator factor H (FH). To escape killing, B. burgdorferi produces an outer surface protein CspZ that binds FH to inhibit complement activation on the cell surface. Immunization with CspZ alone does not protect mice from infection, which we speculate is because FH-binding cloaks potentially protective epitopes. We modified CspZ by conjugating to virus-like particles (VLP-CspZ) and eliminating FH binding (modified VLP-CspZ) to increase immunogenicity. We observed greater bactericidal antibody titers in mice vaccinated with modified VLP-CspZ: A serum dilution of 1:395 (modified VLP-CspZ) vs 1:143 (VLP-CspZ) yielded 50% borreliacidal activity. Immunizing mice with modified VLP-CspZ cleared spirochete infection, as did passive transfer of elicited antibodies. This work developed a novel Lyme disease vaccine candidate by conjugating CspZ to VLP and eliminating FH-binding ability. Such a strategy of conjugating an antigen to a VLP and eliminating binding to the target ligand can serve as a general model for developing vaccines against other bacterial infectious agents.

Structure of AP205 Coat Protein Reveals Circular Permutation in ssRNA Bacteriophages.

AP205 is a single-stranded RNA bacteriophage that has a coat protein sequence not similar to any other known single-stranded RNA phage. Here, we report an atomic-resolution model of the AP205 virus-like particle based on a crystal structure of an unassembled coat protein dimer and a cryo-electron microscopy reconstruction of the assembled particle, together with secondary structure information from site-specific solid-state NMR data. The AP205 coat protein dimer adopts the conserved Leviviridae coat protein fold except for the N-terminal region, which forms a beta-hairpin in the other known single-stranded RNA phages. AP205 has a similar structure at the same location formed by N- and C-terminal beta-strands, making it a circular permutant compared to the other coat proteins. The permutation moves the coat protein termini to the most surface-exposed part of the assembled particle, which explains its increased tolerance to long N- and C-terminal fusions.

Yeast-expressed bacteriophage-like particles for the packaging of nanomaterials.

Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qß, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8 mg from 1 g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.

Different binding modes of free and carrier-protein-coupled nicotine in a human monoclonal antibody.

Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 Å resolution. Nicotine is almost completely (>99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge-charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation-π interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine-carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine-carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine-linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180°. This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained.

The structure of bacteriophage phiCb5 reveals a role of the RNA genome and metal ions in particle stability and assembly.

The structure of the Leviviridae bacteriophage phiCb5 virus-like particle has been determined at 2.9 A resolution and the structure of the native bacteriophage phiCb5 at 3.6 A. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the phiCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, phiCb5 particles are destabilized in solution with elevated salt concentration. The model of the phiCb5 capsid provides an explanation of the salt-induced destabilization of phiCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions. Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the phiCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for phiCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The phiCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.