Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans.In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope protein was used to engineer a next-generation antibody with 5- to 8-fold increased potency in vitro. When administered to nonhuman primates, this antibody conferred protection at a 5-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate the therapeutic potential of antibodies and their in vivo effectiveness.
ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the “out” position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections. IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.
Abstract The SARS-CoV-2 pandemic rages on with devasting consequences on human lives and the global economy 1,2 . The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this novel coronavirus. Here we report the isolation of 61 SARS-CoV-2-neutralizing monoclonal antibodies from 5 infected patients hospitalized with severe disease. Among these are 19 antibodies that potently neutralized the authentic SARS-CoV-2 in vitro , 9 of which exhibited exquisite potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng/mL. Epitope mapping showed this collection of 19 antibodies to be about equally divided between those directed to the receptor-binding domain (RBD) and those to the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that are overlapping with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody targeting RBD, a second targeting NTD, and a third bridging two separate RBDs revealed recognition of the closed, “all RBD-down” conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.
The ars operon of plasmid R773 encodes an As(III)/Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. Tryptophan fluorescence increased upon addition of ATP, ADP, or a nonhydrolyzable ATP analogue. Mg2+ and Sb(III) produced rapid quenching of fluorescence with ADP, no quenching with a nonhydrolyzable analogue, and slow quenching with ATP. The results suggest that slow quenching with ATP reflects hydrolysis of ATP to ADP in the A2 nucleotide-binding site. In an A2 nucleotide-binding site mutant, nucleotides had no effect. In contrast, in an A1 nucleotide-binding mutant, nucleotides still increased fluorescence, but there was no quenching with Mg2+ and Sb(III). This suggests that the A2 site hydrolyzes ATP only when Sb(III) or As(III) is present and when the A1 nucleotide-binding domain is functional. These results support previous hypotheses in which only the A1 nucleotide-binding domain hydrolyzes ATP in the absence of activator (unisite catalysis), and both the A1 and A2 sites hydrolyze ATP when activated (multisite catalysis). The ars operon of plasmid R773 encodes an As(III)/Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. Tryptophan fluorescence increased upon addition of ATP, ADP, or a nonhydrolyzable ATP analogue. Mg2+ and Sb(III) produced rapid quenching of fluorescence with ADP, no quenching with a nonhydrolyzable analogue, and slow quenching with ATP. The results suggest that slow quenching with ATP reflects hydrolysis of ATP to ADP in the A2 nucleotide-binding site. In an A2 nucleotide-binding site mutant, nucleotides had no effect. In contrast, in an A1 nucleotide-binding mutant, nucleotides still increased fluorescence, but there was no quenching with Mg2+ and Sb(III). This suggests that the A2 site hydrolyzes ATP only when Sb(III) or As(III) is present and when the A1 nucleotide-binding domain is functional. These results support previous hypotheses in which only the A1 nucleotide-binding domain hydrolyzes ATP in the absence of activator (unisite catalysis), and both the A1 and A2 sites hydrolyze ATP when activated (multisite catalysis). nucleotide-binding domain adenosine-5′-O-(thiotriphosphate) 4-morpholinepropanesulfonic acid The ars operon of R-factor R773 encodes an arsenite extrusion system that confers resistance in Escherichia coli to the metalloids arsenite (As(III)) and antimonite (Sb(III)) (1Gatti D. Mitra B. Rosen B.P. J. Biol. Chem. 2000; 275: 34009-34012Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This efflux pump has a catalytic subunit, the ArsA ATPase, and a membrane subunit, the ArsB arsenite carrier (2Rosen B.P. Bhattacharjee H. Zhou T. Walmsley A.R. Biochim. Biophys. Acta. 1999; 1461: 207-215Crossref PubMed Scopus (49) Google Scholar, 3Bhattacharjee H. Zhou T., Li, J. Gatti D.L. Walmsley A.R. Rosen B.P. Biochem. Soc. Trans. 2000; 28: 520-526Crossref PubMed Google Scholar). ArsA has N-terminal (A1) and C-terminal (A2) halves that are homologous to each other, most likely as the result of ancestral gene duplication and fusion (4Chen C.M. Misra T.K. Silver S. Rosen B.P. J. Biol. Chem. 1986; 261: 15030-15038Abstract Full Text PDF PubMed Google Scholar). The enzyme has two nucleotide-binding domains, NBD11 and NBD2, both of which are composed of residues from both the A1 and A2 halves (5Zhou T. Radaev S. Rosen B.P. Gatti D.L. EMBO J. 2000; 19: 1-8Crossref PubMed Scopus (212) Google Scholar). Both NBDs are required for metalloid resistance (6Karkaria C.E. Chen C.M. Rosen B.P. J. Biol. Chem. 1990; 265: 7832-7836Abstract Full Text PDF PubMed Google Scholar, 7Kaur P. Rosen B.P. J. Biol. Chem. 1992; 267: 19272-19277Abstract Full Text PDF PubMed Google Scholar). Other pumps with multiple NBDs that exhibit multisite catalysis such as F-type ATPases and ATP-binding cassette ATPases have been proposed to have mechanisms that involve catalytic alternation between the NBDs (where only one is active at a time) and a high degree of cooperativity between the sites (8Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (397) Google Scholar, 9Senior A.E. Acta Physiol. Scand. Suppl. 1998; 643: 213-218PubMed Google Scholar). Kaur (10Kaur P. J. Biol. Chem. 1999; 274: 25849-25854Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) has suggested that ArsA exhibits both unisite and multisite catalysis in which only NBD1 participates in unisite catalysis and that a functional A1 NBD is required for NBD2 to participate in multisite catalysis. We have considered a similar alternating site model for the function of ArsA, where the two NBDs alternate between open and closed conformations in a concerted and interactive manner, coupling the energy of ATP hydrolysis to the transfer of As(III) or Sb(III) at the metal site of ArsA to the ArsB carrier (11Zhou T. Radaev S. Rosen B.P. Gatti D.L. J. Biol. Chem. 2001; 276: 30414-30422Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In the absence of metalloid, ArsA catalyzes hydrolysis of ATP at a low basal rate (k = 0.001 s−1) (13Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 1999; 274: 16153-16161Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Under presteady state conditions the addition of Sb(III) produces two bursts of phosphate liberation, one of which is ∼250-fold faster than the other (k = 49 and 0.2 s−1) (12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). From these results, it appears that both NBDs hydrolyze ATP in the activated state. However, the two NBDs are not equivalent in either structure (5Zhou T. Radaev S. Rosen B.P. Gatti D.L. EMBO J. 2000; 19: 1-8Crossref PubMed Scopus (212) Google Scholar, 11Zhou T. Radaev S. Rosen B.P. Gatti D.L. J. Biol. Chem. 2001; 276: 30414-30422Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) or catalytic rates (12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), which raises the question of whether they are functionally equivalent or whether they play different roles in metalloid resistance. To examine the role of the individual NBDs, we previously constructed two single tryptophan ArsAs (F141W and W159) that report nucleotide occupancy and hydrolysis in NBD1 (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Zhou T. Liu S. Rosen B.P. Biochemistry. 1995; 34: 13622-13626Crossref PubMed Scopus (24) Google Scholar). In each half there is a 12-residue sequence (DTAPTGH) that is found in all ArsA homologues from bacteria to humans (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In the ArsA structure these are seen as extended regions that connect the single regulatory metalloid-binding domain with the two NBDs and probably function in signal transduction between the two substrate sites and the regulatory site (5Zhou T. Radaev S. Rosen B.P. Gatti D.L. EMBO J. 2000; 19: 1-8Crossref PubMed Scopus (212) Google Scholar). Trp141 is located at the N-terminal end of the A1142DTAPTGH148, whereas Trp159 is near the C-terminal end. Asp142 is a Mg2+ligand in NBD, and His148 is an Sb(III) ligand in the metalloid binding site (5Zhou T. Radaev S. Rosen B.P. Gatti D.L. EMBO J. 2000; 19: 1-8Crossref PubMed Scopus (212) Google Scholar, 16Bhattacharjee H. Rosen B.P. Biometals. 2000; 13: 281-288Crossref PubMed Scopus (12) Google Scholar). Trp141 primarily reports binding of MgADP, but indirectly reports ATP hydrolysis by the filling NBD1 with the product ADP (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Zhou T. Liu S. Rosen B.P. Biochemistry. 1995; 34: 13622-13626Crossref PubMed Scopus (24) Google Scholar). Trp159 reports conformational changes in the vicinity of NBD1 during hydrolysis of ATP (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 15Zhou T. Liu S. Rosen B.P. Biochemistry. 1995; 34: 13622-13626Crossref PubMed Scopus (24) Google Scholar). The response of spectroscopic signals to nucleotide binding and hydrolysis at NBD1 has allowed a detailed analysis of the catalytic cycle of ArsA (12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 13Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 1999; 274: 16153-16161Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 17Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. Biochem. J. 2001; 360: 589-597Crossref PubMed Google Scholar). In this study we report the properties of M446W, a single tryptophan derivative of ArsA. Trp446 is adjacent to the A2447DTAPTGH sequence and is in the vicinity of NBD2. Trp446 occupies the equivalent position in A2 that Trp141 does in A1. In the absence of metalloid (unisite conditions), both ADP and ATP produced an enhancement of M446W protein fluorescence that was stable with time, indicating binding but not hydrolysis of ATP in NBD2. When the enzyme was preincubated with Sb(III) and Mg2+ (multisite conditions), ADP produced a rapid quenching of fluorescence. Addition of nonhydrolyzable ATP analogues resulted in a stable enhancement of fluorescence. In contrast, ATP rapidly enhanced fluorescence followed by a slow quenching to the level with ADP, consistent with ATP hydrolysis under multisite conditions. Thus, although Trp141 and Trp446 occupy equivalent positions in the A1 and A2 halves of ArsA, respectively, their fluorescent responses to nucleotide binding and hydrolysis differ considerably, indicating nonequivalence of NBD1 and NBD2. These results support the hypothesis that both NBD1 and NBD2 hydrolyze ATP under multisite conditions but that only NBD1 and not NBD2 participates in unisite catalysis (10Kaur P. J. Biol. Chem. 1999; 274: 25849-25854Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). All of the E. coli strains and plasmids used in this study are listed in Table I. Cells were grown in Luria-Bertani medium (18Miller J. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, New York1992Google Scholar) at 37 °C. Ampicillin (125 μg/ml) and tetracycline (12.5 μg/ml) were added as required. ATPγS was purchased from Sigma.Table IStrains and plasmidsStrain/PlasmidGenotype/descriptionRef. E. coli strains ES1301mutS lacZ53 mutS201::Tn5 thyA36 rha-5 metB1 deoC IN(rrnD-rrnE)Promega JM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Δ(lac-proAB) F′ [traD36 proA+B+laclqlacZΔM15](19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, New York1989Google Scholar)plasmids pTZ4H6 arsA gene with all 4 tryptophan codons mutated to those of tyrosines and six histidine codons added to 3′-end, Tcr(14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) pTZ4H6M446WSite-directed mutagenesis of codon 446 to Trp codon in arsA gene of pTZ4H6, AprThis study pTZ4H6M446WG18RSite-directed mutagenesis of codon 18 to Arg codon in arsA gene of pTZ4H6M446W, TcrThis study pTZ4H6M446WG337RSite-directed mutagenesis of codon 337 to Arg codon in arsA gene of pTZ4H6M446W, TcrThis study pTZ4H6M446WG18RG337RSite-directed mutagenesis of codons 18 and 337 to Arg codons in arsA gene of pTZ4H6M446W, TcrThis study Open table in a new tab Plasmid isolation, DNA restriction endonuclease analysis, ligation, and transformation were performed by standard methods (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, New York1989Google Scholar). Restriction enzymes and nucleic acid-modifying enzymes were purchased from Invitrogen. WizardTMplus minipreps DNA purification system and WizardTM DNA clean-up system (Promega) were used to prepare plasmid DNA for restriction enzyme digestion and to recover DNA fragments from low melting agarose gels, respectively. Mutations in the arsAgene were introduced by site-directed mutagenesis using the Altered SitesTM in vitro mutagenesis system (Promega) with plasmid pTZ4H6, which contains the arsA andarsB genes with the sequence for six histidine codons added at the 3′ end of the arsA gene. The arsAgene in pTZ4H6, which had previously been mutated to remove the four native tryptophan codons, was used as the template to produce the M446WH6 arsA mutant (or for simplicity, M446W). G18R and G337R substitutions were then introduced into M446W ArsA to obtain M446W G18R, M446W G337R, and M446W G18R/G337R mutants. The mutagenic oligonucleotides used and the respective changes (underlined) are as follows: M446W, 5′-GGAGCCGTATCCCACACCACGAAACGT-3′; G18R, 5′-TTACCCACGCGTCCTTTACCCG-3′; G337R, 5′-TTCCCCACGCGACCTTTACCC-3′. All mutations were confirmed by sequencing the entire arsA gene using a Cy5-AUTOREAD sequencing kit with an ALFexpress system (Amersham Biosciences). Plasmid DNA for sequencing was prepared with a miniprep kit (Qiagen). JM109 cells harboring the indicated plasmids were grown at 37 °C in Luria-Bertani medium toA 600 nm = 0.6–0.8, at which point 0.1 mm isopropyl β-d-thiogalactopyranoside was added to induce arsA expression. After another 2.5 h of growth, the cells were harvested by centrifugation. Soluble ArsA was purified as described (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). ATPase activity was measured using an NADH-coupled assay (20Vogel G. Steinhart R. Biochemistry. 1976; 15: 208-216Crossref PubMed Scopus (206) Google Scholar) with 5 mm ATP and 2.5 mm MgCl2 unless otherwise noted. Fluorescence measurements were performed using an SLM-8000C spectrofluorometer with a built-in magnetic stirrer. The bandwidths for emission and excitation monochromators were 4 nm. Tryptophan fluorescence was monitored with an excitation wavelength of 295 nm and an emission wavelength of 331 nm. The fluorescence of the assay buffer (50 mm MOPS-KOH, pH 7.5) alone was subtracted from each spectrum. ATP, ADP, ATPγS, MgCl2, and antimonite (in the form of potassium antimonyl tartrate) were added as indicated. The concentration of ArsA was 1.25 μm in all assays. The structures of the M446W ArsA with MgATP and antimonite or MgADP and antimonite were modeled on the crystal structures of the wild type enzyme with the same ligands (11Zhou T. Radaev S. Rosen B.P. Gatti D.L. J. Biol. Chem. 2001; 276: 30414-30422Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Molecular modeling with energy minimization was performed using the program MODELLER (21Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10783) Google Scholar). Beginning with a derivative of ArsA in which all of the tryptophan residues had been replaced with tyrosine residues and a six-histidine tag had been added to the C terminus (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), Met446 was changed to a tryptophan residue. Cells expressing the mutated arsA gene were as resistant to arsenite as cells expressing a wild type arsA gene (data not shown). Purified M446W ArsA exhibited basal ATPase activity of 82 nmol/mg/min and Sb(III)-stimulated ATPase activity of 327 nmol/mg/min. This is comparable to the activity of the tryptophan-free histidine-tagged ArsA (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The intrinsic protein fluorescence of the M446W ArsA was analyzed (Fig.1). M446W denatured with 8 murea had a fluorescence emission spectrum indistinguishable from an equimolar amount of free tryptophan, with a λmax of 354 nm. Native M446W exhibited a substantial blue shift (λmax= 328 nm) and fluorescence enhancement. Addition of Sb(III) and saturating MgATP (1 mm) increased the fluorescence, and Sb(III) and 1 mm MgADP quenched the fluorescence. Sb(III) alone had no effect (data not shown). There was no shift in wavelength associated with the addition of those ligands, suggesting that the polarity of the environment of the indole side chain of Trp446 was not greatly altered. To examine the structural basis of fluorescence changes in the ATP- and ADP-bound forms, the position of the tryptophan residue under both sets of conditions was modeled on the known crystal structures of the wild type enzyme with the same ligands (11Zhou T. Radaev S. Rosen B.P. Gatti D.L. J. Biol. Chem. 2001; 276: 30414-30422Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Because the structure of nucleotide-free ArsA has not been determined, the differences between the nucleotide-bound and nucleotide-free enzymes cannot be modeled. In the ADP-bound form (Fig. 2 A) Trp446 is oriented toward the bound nucleotide compared with the ATP-bound form (Fig. 2 B). This brings the indole ring closer to the phenyl ring of Phe432 (Fig.2 C). In the ATP-bound form, the imidazole –NH of Trp446 is ∼6 Å at its closest approach to the Phe432 phenyl ring. In the ADP-bound form the two aromatic rings are brought closer together (nearly perpendicular) so that the imidazole –NH comes within 3 Å of the phenyl ring. At this distance an indole amino proton can form a hydrogen bond with a phenyl ring that can quench tryptophan fluorescence (22Rouviere N. Vincent M. Craescu C.T. Gallay J. Biochemistry. 1997; 36: 7339-7352Crossref PubMed Scopus (53) Google Scholar). This suggests that the difference in fluorescence between the ATP- and ADP-bound forms is related to the relationship of the indole ring of Trp446 to the phenyl ring of Phe432. The time dependence of the nucleotide responses under multisite conditions (in the presence of Sb(III)) was examined in more detail. The fluorescence quenching by MgADP observed in Fig. 1was rapid; in the presence of Sb(III), addition of 50 μmMgADP to M446W produced a quick decrease in fluorescence intensity (Fig. 3, curve 5). In contrast, either 50 μm or 1 mm MgATP in the presence of Sb(III) produced a rapid rise in fluorescence followed by a slower quenching (Fig. 3, curves 3 and 4). With extended incubation time with 50 μm ATP, the fluorescence decreased to the same level as with MgADP, and the rate of quenching paralleled the rate of ATP hydrolysis (data not shown). Note that the concentration of ATP in curve 4 is only 5% of that in Fig. 1 so that ATP is depleted during the course of this assay but not in the duration of the assay shown in Fig. 1. The simplest interpretation of this result is that the decay of fluorescence represents the change in occupancy of the A2 NBD from the substrate ATP to the product ADP during the course of hydrolysis. Consistent with this idea, addition of the nonhydrolyzable ATP analog ATPγS also induced enhancement of fluorescence, but no decay was observed even with prolonged incubation (Fig. 3, curve 1). When added with MgATP, beryllium fluoride (BeFx), which traps ATPases in a conformation resembling the transition state (23Sankaran B. Bhagat S. Senior A.E. Biochemistry. 1997; 36: 6847-6853Crossref PubMed Scopus (62) Google Scholar), also produced an enhancement of fluorescence with no subsequent decay of the fluorescence signal (Fig. 3,curve 2). In the absence of antimonite ArsA hydrolyzes ATP at a low basal rate, which has been attributed to unisite hydrolysis in NBD1 (10Kaur P. J. Biol. Chem. 1999; 274: 25849-25854Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Consistent with this hypothesis, addition of either 1 mm or 50 μm MgATP produced a fluorescence enhancement of the M446W ArsA without subsequent decay of fluorescence even after extended times (Fig.4, curves 1 and 2). This suggests that ATP is bound in NBD2 but not hydrolyzed under unisite conditions. Under these conditions MgADP produced a fluorescence enhancement rather than quenching, although this was to a lesser extent than ATP (Fig. 4, curve 3). The transition from unisite to multisite conditions was examined by sequential addition of nucleotide, Mg2+, and Sb(III) (Fig.5). There was fluorescence enhancement following addition of ATP (Fig. 5, curve 1), ATPγS (Fig.5, curve 2), or ADP (Fig. 5, curve 3). Subsequent addition of Mg2+ partially reversed the fluorescence enhancement, which could result from a conformational change in the local environment of NBD2 when Mg2+ is bound. Multisite conditions were initiated by the addition of Sb(III). As when the protein was preincubated with Sb(III), this resulted in a rapid quenching by ADP (Fig. 5, curve 3) and a slow quenching by ATP (Fig. 5, curve 1). The ATP quenching most likely reflects hydrolysis in NBD2, since there was no slow quench with ATPγS but rather a further enhancement of fluorescence (Fig. 5,curve 2). What is the requirement for two functional NBDs to produce these nucleotide-dependent effects on Trp446fluorescence? To examine this question, mutations in the P-loops of the two NBDs were combined with the M446W mutation individually and together. The G18R and G337R mutations have been shown to result in loss of resistance to arsenicals and loss of nucleotide binding in the A1 or A2 NBD, respectively (6Karkaria C.E. Chen C.M. Rosen B.P. J. Biol. Chem. 1990; 265: 7832-7836Abstract Full Text PDF PubMed Google Scholar, 7Kaur P. Rosen B.P. J. Biol. Chem. 1992; 267: 19272-19277Abstract Full Text PDF PubMed Google Scholar, 24Karkaria C.E. Rosen B.P. Arch. Biochem. Biophys. 1991; 288: 107-111Crossref PubMed Scopus (21) Google Scholar). In M446W into which the G337R mutation was introduced there was no response of nucleotide under either unisite or multisite conditions (Fig.6 A). However, with M446W G18R, addition of ATP (Fig. 6 B, curve 1), ATPγS (Fig.6 B, curve 2), or ADP (Fig. 6 B,curve 3) under unisite conditions gave the same response as the wild type (Fig. 5). Addition of Sb(III) to initiate multisite conditions produced a small quench that may be caused by a conformational change upon Sb(III) binding, but there was no evidence of ATP hydrolysis by a slow quenching with ATP. This result suggests that NBD2 can bind nucleotides in the absence of a functional NBD1 but that it is locked into a conformation that prevents subsequent steps in the catalytic cycle. Many types of transport ATPases are multisubunit complexes that contain two or more nucleotide-binding sites. The ways in which these sites interact with each other and participate in catalysis are still incompletely understood for even the best characterized proteins. For example, F-type ATPases have two classes of NBDs, both catalytic and noncatalytic (25Cross R.L. Biochim. Biophys. Acta. 2000; 1458: 270-275Crossref PubMed Scopus (63) Google Scholar). To explain the basis of the strong interactions between the catalytic NBDs in F1, Boyer and co-workers (26Kayalar C. Rosing J. Boyer P.D. J. Biol. Chem. 1977; 252: 2486-2491Abstract Full Text PDF PubMed Google Scholar) proposed a binding change mechanism involving an alternation of catalytic sites, which took two decades of intensive study in many laboratories to be verified. Subsequently the ATP-binding cassette-type drug transporter P-glycoprotein has been suggested to utilize a similar alternating site mechanism (9Senior A.E. Acta Physiol. Scand. Suppl. 1998; 643: 213-218PubMed Google Scholar, 27Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1948) Google Scholar). The ArsAB As(III)/Sb(III)-translocating ATPase is a resistance pump that catalyzes efflux of toxic metalloid salts (1Gatti D. Mitra B. Rosen B.P. J. Biol. Chem. 2000; 275: 34009-34012Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The catalytic subunit, ArsA, has two interacting NBDs (2Rosen B.P. Bhattacharjee H. Zhou T. Walmsley A.R. Biochim. Biophys. Acta. 1999; 1461: 207-215Crossref PubMed Scopus (49) Google Scholar). In the absence of Sb(III) or As(III) the enzyme hydrolyzes ATP at a slow basal rate, which seems to be catalyzed by only NBD1 (unisite catalysis), whereas in the presence of metalloid salts both sites are catalytic (10Kaur P. J. Biol. Chem. 1999; 274: 25849-25854Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Under presteady state conditions in the presence of Sb(III) (multisite conditions), one NBD hydrolyzes ATP 250-fold faster than the other (12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). From the rate of fluorescence quenching in the single tryptophan derivatives of ArsA that have tryptophan residues near NBD1 (Trp141 and Trp159), the fast site can be identified as NBD1, which identifies the slow site as NBD2. The fluorescent properties of the F141W and W159 single tryptophan-containing ArsAs have been extremely informative about the catalytic properties of NBD1. In particular, under unisite conditions both Trp141 and Trp159 report slow hydrolysis (12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), whereas under multisite conditions Trp159 reports rapid conformational changes associated with ATP hydrolysis (13Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 1999; 274: 16153-16161Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). From these results it is clear that NBD1 participates in both unisite and multisite conditions. From structure-based alignment of the A1 and A2 halves of ArsA, Met446 is identified as the residue in A2 that is equivalent to Phe141 in A1 (11Zhou T. Radaev S. Rosen B.P. Gatti D.L. J. Biol. Chem. 2001; 276: 30414-30422Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To examine the properties of NBD2, Met446 was changed to a tryptophan residue in a tryptophan-free background. The fluorescent responses of M446W ArsA are quite different from those of the F141W or W159 proteins. Both F141W and W159 ArsAs exhibit quenching of fluorescence with ATP that parallels the unisite rate of hydrolysis (14Zhou T. Rosen B.P. J. Biol. Chem. 1997; 272: 19731-19737Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In contrast, in the absence of Sb(III), ATP produces a stable enhancement of fluorescence of M446W, indicating that NBD2 does not hydrolyze ATP under unisite conditions. On the other hand, under multisite conditions W159 ArsA exhibits a quenching of fluorescence that is much more rapid than the steady state rate of hydrolysis. The interpretation of these results is that the highly fluorescent form of W159 under unisite conditions is because of buildup in the steady state of a conformational intermediate that is in slow equilibrium with the ground state of the enzyme (13Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 1999; 274: 16153-16161Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Under multisite conditions isomerization of the two forms is much faster such that a prior step in the catalytic cycle such as product release becomes rate-limiting. In contrast, the quenching rate of M446W fluorescence in the presence of Sb(III) is close to the steady state rate of hydrolysis, indicating that NBD2 is catalytic under multisite conditions. From their evolutionary relationships and overall structural similarities, it is tempting to speculate that NBD1 and NBD2 have equivalent roles in ArsA function. In the crystal structure in which both are filled with MgADP, NBD1 is occluded while NBD2 is open (5Zhou T. Radaev S. Rosen B.P. Gatti D.L. EMBO J. 2000; 19: 1-8Crossref PubMed Scopus (212) Google Scholar). The occluded nature of NBD1 prevents exchange of ADP with other nucleotides, while the open NBD2 allows facile exchange with ATP or ATP analogues (11Zhou T. Radaev S. Rosen B.P. Gatti D.L. J. Biol. Chem. 2001; 276: 30414-30422Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Yet the occlusion in the crystal structure may result from capturing the enzyme in one particular conformation that represents one step in the catalytic cycle, and the two sites may be symmetric at other times during catalysis. These considerations have led to the alternating site models for ArsA (10Kaur P. J. Biol. Chem. 1999; 274: 25849-25854Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 11Zhou T. Radaev S. Rosen B.P. Gatti D.L. J. Biol. Chem. 2001; 276: 30414-30422Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). On the other hand, it is possible that the two sites have different roles in ArsA function. For example, under multisite conditions the role of NBD1 may be to activate NBD2, and perhaps hydrolysis only at NBD2 is coupled to transport. Although the results of this study do not distinguish between different models, they demonstrate that the two nucleotide-binding sites have intrinsic differences. This gives more weight to the possibility that the two NBDs have dissimilar functional roles in catalysis.
Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy (cryo-EM) analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species.
Serum characterization and antibody isolation are transforming our understanding of the humoral immune response to viral infection. Here, we show that epitope specificities of HIV-1-neutralizing antibodies in serum can be elucidated from the serum pattern of neutralization against a diverse panel of HIV-1 isolates. We determined "neutralization fingerprints" for 30 neutralizing antibodies on a panel of 34 diverse HIV-1 strains and showed that similarity in neutralization fingerprint correlated with similarity in epitope. We used these fingerprints to delineate specificities of polyclonal sera from 24 HIV-1-infected donors and a chimeric siman-human immunodeficiency virus-infected macaque. Delineated specificities matched published specificities and were further confirmed by antibody isolation for two sera. Patterns of virus-isolate neutralization can thus afford a detailed epitope-specific understanding of neutralizing-antibody responses to viral infection.
