Biological and Structural Evaluation of 10<i>R</i>- and 10<i>S</i>‑Methylthio-DDACTHF Reveals a New Role for Sulfur in Inhibition of Glycinamide Ribonucleotide Transformylase

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the <i>de novo</i> purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10<i>S</i>- and 10<i>R</i>-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (<i>K</i>_i = 210 nM for 10<i>R</i>, and<i> K</i>_i = 180 nM for 10<i>S</i>) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC₅₀ = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC₅₀ = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. <i>In silico</i> docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza

Crystal structure of the HL4E10 Fab.

<p>(<b>A</b>) Cartoon representation of the superimposition of the two HL4E10 Fab structures in the asymmetric unit. The two HL4E10 Fabs (LH and AB) are shown in dark and light gray, respectively. The CDR loops are color coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g001" target="_blank">Fig. 1</a>&<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g002" target="_blank">2</a>: CDR L1 yellow, CDR L2 cyan, CDR L3 orange, CDR H1 blue, CDR H2 pink, CDR H3 green. The Cα atoms of Fab LH and Fab AB superimpose with an r.m.s.d. of 0.63 Å. (<b>B</b>) Superimposition of the combining sites of HL4E10 Fab LH (CDR loops colored) and Fab AB (CDR loops gray) (in a similar orientation to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g004" target="_blank">Fig. 4C</a>) reveals a rigid assembly without significant conformational differences. (<b>C</b>) Wall-eyed stereo representation of the molecular interactions which rigidify the HL4E10 CDR loops and lock the side chains in conformations predefined for high affinity ligand binding. For example, hydrogen bonds (black), CH-π interactions (grey), and hydrophobic stacking interactions occur at the interface of CDR L3 with CDRs H3, H2, and H1.</p

Structure prediction and automated modeling of HL4E10.

<p>Cartoon representation of the superimposition of the experimentally determined HL4E10 heavy and light chain variable domain structures and the three top scoring heavy (1W72, 2G75, 1ADQ) and light chain (1A7P, 1GIG, and 1DL7) computational models. Heavy and light chains are shown in dark and light gray, respectively. The CDR loops of HL4E10 are color coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g001" target="_blank">Fig. 1</a>&<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g002" target="_blank">2</a>: CDR L1 yellow, CDR L2 cyan, CDR L3 orange, CDR H1 blue, CDR H2 pink, CDR H3 green. The Cα atoms of the experimentally determined HL4E10 structure and the computational models superimpose well, with an average r.m.s.d. of 0.68 Å for the heavy chains and 0.77 Å for the light chains, respectively. The largest deviations are observed, as expected, in the CDR loops, namely L1, L3 and H3.</p

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza

Amino-acid sequence alignment of the HL4E10 hamster IgG heavy chain with those of other hamster antibodies.

<p>The HL4E10 heavy chain is aligned with H28.710 (U17166 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Collins1" target="_blank">[33]</a>), 145.2c11 (U17871 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Alegre1" target="_blank">[31]</a>), and 3A5-1 (S80616 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone.0019828-Mallender1" target="_blank">[30]</a>). Color coding and shading is used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019828#pone-0019828-g001" target="_blank">Fig. 1</a>, CDR H1 is in blue, CDR H2 in pink, CDR H3 in green, and the glycine-, proline-, cysteine-rich hinge region between V_HC_H1 and C_H2C_H3 is shaded gray.</p

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza