Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ...ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 Å resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 Å root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 Å) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides α-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.
Baculovirus RNA 5′-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we ...report the crystal structure of BVP in a phosphate-bound state at 1.5 Å resolution. BVP adopts the characteristic cysteine-phosphatase α/β fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif 118HCTHGXNRT126 in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.
Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of ...protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire—pre- and post-vaccination—and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.
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•Serum vaccine response is dominated by a small number of abundant antibody clonotypes•Vaccine-boosted antibodies predominantly target conserved norovirus epitopes•Identified cross-genogroup and strain-specific epitopes•Discovered a pandemic-genotype neutralizing antibody recognizing a conserved epitope
Human norovirus (HuNoV) is a leading cause of gastroenteritis. Lindesmith et al. identify circulating serum antibodies following experimental HuNoV vaccination in humans and map them to viral epitopes. One antibody recognizes a neutralizing epitope conserved across three decades of pandemic strains and neutralizes virus in vitro, demonstrating that vaccination can elicit pandemic-strain neutralizing antibody responses in some individuals.
Mismatch repair (MMR) ensures the fidelity of DNA replication, initiates the cellular response to certain classes of DNA damage, and has been implicated in the generation of immune diversity. Each of ...these functions depends on MutSα (MSH2•MSH6 heterodimer). Inactivation of this protein complex is responsible for tumor development in about half of known hereditary nonpolyposis colorectal cancer kindreds and also occurs in sporadic tumors in a variety of tissues. Here, we describe a series of crystal structures of human MutSα bound to different DNA substrates, each known to elicit one of the diverse biological responses of the MMR pathway. All lesions are recognized in a similar manner, indicating that diversity of MutSα-dependent responses to DNA lesions is generated in events downstream of this lesion recognition step. This study also allows rigorous mapping of cancer-causing mutations and furthermore suggests structural pathways for allosteric communication between different regions within the heterodimer.
•Purification of clade A and C HIV-1 envelope (Env) trimers by a novel non-affinity method.•Env trimers produced in pre-fusion closed conformation without antibody capture.•Non-affinity purified ...trimers show minimal exposure of CD4-induced or V3 epitopes.•Scalable process yields up to 0.2 g/L of purified prefusion-closed HIV-1-Env trimer.
Metastable glycosylated immunogens present challenges for GMP manufacturing. The HIV-1 envelope (Env) glycoprotein trimer is covered by N-linked glycan comprising half its mass and requires both trimer assembly and subunit cleavage to fold into a prefusion-closed conformation. This conformation, the vaccine-desired antigenic state, is both metastable to structural rearrangement and labile to subunit dissociation. Prior reported GMP manufacturing for a soluble trimer stabilized in a near-native state by disulfide (SOS) and Ile-to-Pro (IP) mutations has employed affinity methods based on antibody 2G12, which recognizes only ~30% of circulating HIV strains. Here, we develop a scalable manufacturing process based on commercially available, non-affinity resins, and we apply the process to current GMP (cGMP) production of trimers from clades A and C, which have been found to boost cross-clade neutralizing responses in vaccine-test species. The clade A trimer, which we named “BG505 DS-SOSIP.664”, contained an engineered disulfide (201C-433C; DS) within gp120, which further stabilized this trimer in a prefusion-closed conformation resistant to CD4-induced triggering. BG505 DS-SOSIP.664 was expressed in a CHO-DG44 stable cell line and purified with initial and final tangential flow filtration steps, three commercially available resin-based chromatography steps, and two orthogonal viral clearance steps. The non-affinity purification enabled efficient scale-up, with a 250 L-scale cGMP run yielding 9.6 g of purified BG505 DS-SOSIP.664. Antigenic analysis indicated retention of a prefusion-closed conformation, including recognition by apex-directed and fusion peptide-directed antibodies. The developed manufacturing process was suitable for 50 L-scale production of a second prefusion-stabilized Env trimer vaccine candidate, ConC-FP8v2 RnS-3mut-2G-SOSIP.664, yielding 7.8 g of this consensus clade C trimer. The successful process development and purification scale-up of HIV-1 Env trimers from different clades by using commercially available materials provide experimental demonstration for cGMP manufacturing of trimeric HIV-Env vaccine immunogens, in an antigenically desired conformation, without the use of costly affinity resins.
HIV-1 envelope (Env) trimers, stabilized in a prefusion-closed conformation, can elicit humoral responses capable of neutralizing HIV-1 strains closely matched in sequence to the immunizing strain. ...One strategy to increase elicited neutralization breadth involves vaccine priming of immune responses against a target site of vulnerability, followed by vaccine boosting of these responses with prefusion-closed Env trimers. This strategy has succeeded at the fusion peptide (FP) site of vulnerability in eliciting cross-clade neutralizing responses in standard vaccine-test animals. However, the breadth and potency of the elicited responses have been less than optimal. Here, we identify three mutations (3mut), Met302, Leu320, and Pro329, that stabilize the apex of the Env trimer in a prefusion-closed conformation and show antigenically, structurally, and immunogenically that combining 3mut with other approaches (e.g., repair and stabilize and glycine-helix breaking) yields well-behaved clade C-Env trimers capable of boosting the breadth of FP-directed responses. Crystal structures of these trimers confirmed prefusion-closed apexes stabilized by hydrophobic patches contributed by Met302 and Leu320, with Pro329 assuming canonically restricted dihedral angles. We substituted the N-terminal eight residues of FP (FP8, residues 512 to 519) of these trimers with the second most prevalent FP8 sequence (FP8v2, AVGLGAVF) and observed a 3mut-stabilized consensus clade C-Env trimer with FP8v2 to boost the breadth elicited in guinea pigs of FP-directed responses induced by immunogens containing the most prevalent FP8 sequence (FP8v1, AVGIGAVF). Overall, 3mut can stabilize the Env trimer apex, and the resultant apex-stabilized Env trimers can be used to expand the neutralization breadth elicited against the FP site of vulnerability.
A major hurdle to the development of an effective HIV-1 vaccine is the elicitation of serum responses capable of neutralizing circulating strains of HIV, which are extraordinarily diverse in sequence and often highly neutralization resistant. Recently, we showed how sera with 20 to 30% neutralization breadth could, nevertheless, be elicited in standard vaccine test animals by priming with the most prevalent N-terminal 8 residues of the HIV-1 fusion peptide (FP8), followed by boosting with a stabilized BG505-envelope (Env) trimer. Here, we show that subsequent boosting with a 3mut-apex-stabilized consensus C-Env trimer, modified to have the second most prevalent FP8 sequence, elicits higher neutralization breadth than that induced by continued boosting with the stabilized BG505-Env trimer. With increased neutralizing breadth elicited by boosting with a heterologous trimer containing the second most prevalent FP8 sequence, the fusion peptide-directed immune-focusing approach moves a step closer toward realizing an effective HIV-1 vaccine regimen.
While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could ...neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses - both X-ray and cryo-EM - revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability.
The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.
The HIV fusion peptide (FP) is a promising vaccine target. FP-directed monoclonal antibodies from vaccinated macaques have been identified that neutralize up to ∼60% of HIV strains; these ...vaccinations, however, have involved ∼1 year with an extended neutralization-eclipse phase without measurable serum neutralization. Here, in 32 macaques, we test seven vaccination regimens, each comprising multiple immunizations of FP-carrier conjugates and HIV envelope (Env) trimers. Comparisons of vaccine regimens reveal FP-carrier conjugates to imprint cross-clade neutralizing responses and a cocktail of FP conjugate and Env trimer to elicit the earliest broad responses. We identify a signature, appearing as early as week 6 and involving the frequency of B cells recognizing both FP and Env trimer, predictive of vaccine-elicited breadth ∼1 year later. Immune monitoring of B cells in response to vaccination can thus enable vaccine insights even in the absence of serum neutralization, here identifying FP imprinting, cocktail approach, and early signature as means to improve FP-directed vaccine responses.
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•Immunization in 32 rhesus macaques reveals FP priming to imprint cross-clade responses•Identifying an early B cell signature predictive of vaccine outcome•Priming with a cocktail of FP and trimer elicits the earliest neutralizing responses•B cell immune monitoring enables vaccine insights, even without serum neutralization
Immune monitoring of B cells in response to vaccination can enable early insights, even in the absence of serum neutralization. Cheng et al. observe an early B cell signature in NHPs predictive of the vaccine outcome, with priming of HIV FP imprinting cross-clade neutralizing FP-directed vaccine responses.
Periplasmic binding proteins comprise a superfamily that is present in archaea, prokaryotes, and eukaryotes. Periplasmic binding protein ligand-binding sites have diversified to bind a wide variety ...of ligands. Characterization of the structural mechanisms by which functional adaptation occurs is key to understanding the evolution of this important protein superfamily. Here we present the structure and ligand-binding properties of a maltotriose-binding protein identified from the Thermus thermophilus genome sequence. We found that this receptor has a high affinity for the trisaccharide maltotriose (Kd<1 μM) but little affinity for disaccharides that are transported by a paralogous maltose transport operon present in T. thermophilus. Comparison of this structure to other proteins that adopt the maltose-binding protein fold but bind monosaccharides, disaccharides, or trisaccharides reveals the presence of four subsites that bind individual glucose ring units. Two loops and three helical segments encode adaptations that control the presence of each subsite by steric blocking or hydrogen bonding. We provide a model in which the energetics of long-range conformational equilibria controls subsite occupancy and ligand binding.
Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we ...report the crystal structure of BVP in a phosphate-bound state at 1.5 {angstrom} resolution. BVP adopts the characteristic cysteine-phosphatase {alpha}/{beta} fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif {sup 118}HCTHGXNRT{sup 126} in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.
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