crossmark LETTER TO THE EDITOR
The Broadly Neutralizing, Anti-HIV Antibody 4E10: an Open and Shut Case? Roland K. Strong, Kathryn A. K. Finton Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
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3274
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FIG 1 (A and B) Views of the two molecules in the asymmetric unit of the crystal structure of the “ligand-free” 4E10 Fab (PDB code 5CIP) (1), shown as cartoon ribbons colored to highlight different substructures. Chunks of neighboring 4E10 Fabs in the crystal lattice (yellow) are shown in a spherical representation. (C) The structure of the complex between the 4E10 Fab and an HIV envelope protein-derived peptide (2FX7) (5) is shown as in panels A and B. In these views, the packing constraints on the 4E10 CDR-H3 loop imposed by the epitope peptide ligand or neighboring Fabs are clearly revealed. (D) The phosphate-binding site in the 4E10 Fab/peptide complex is shown, with the molecular surface of the Fab, colored according to electrostatic potential, shown as a semitransparent overlay.
a phosphate-binding site observed in another 4E10 complex structure (8), where one of two key positively charged side chains bracketing the phosphate is a lysine in the bound peptide (Fig. 1D). However, since 4E10 membrane binding is a property of the ligand-free antibody (3, 6, 10, 11), this site is unlikely to explain this property.
Citation Strong RK, Finton KAK. 2016. The broadly neutralizing, anti-HIV antibody 4E10: an open and shut case? J Virol 90:3274 –3275. doi:10.1128/JVI.02935-15. Editor: F. Kirchhoff Address correspondence to Roland K. Strong, [email protected]. For the author reply, see doi:10.1128/JVI.02970-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Journal of Virology
March 2016 Volume 90 Number 6
Downloaded from http://jvi.asm.org/ on April 15, 2016 by TULANE UNIV
ujas et al. (1) recently studied the recognition mechanism of the anti-HIV antibody 4E10. We completely agree with their overall conclusion, that 4E10 “must recognize an antigenic structure more complex than just the linear ␣-helical epitope,” which echoes our own analysis (2): “4E10 may make contacts to elements of Env outside of the linear 4E10 epitope.” Their ligand-free Fab structure showed “that the CDR-H3 loop does not undergo significant conformational changes during recognition of the epitope,” consistent with thermodynamics that “ruled out major conformational changes of CDR-H3.” However, our structure of the ligand-free 4E10 Fv (3) had revealed a dramatic restructuring of CDR-H3, compared to Fv or Fab structures bound to HIV peptides (4–7). The difference was accounted for by the “low pH required to crystallize the Fv construct” and/or by “different structural stabilities” (1). Crystallization conditions can affect structure, and our ligand-free Fv crystallization conditions were nonphysiological, as noted by others (8). However, these conditions are not “extreme” in terms of protein crystallization overall. Solution thermostabilities of Fvs are also typically lower than those of Fabs (9), partly because Fvs lack the interchain disulfide that stabilizes Fabs. Since we had carefully shown that the 4E10 Fv retains the structure, binding, and neutralization properties of the 4E10 Fab (6), these explanations may not fully account for the apparent discrepancy. Rojas et al. state that the binding thermodynamics also argue against major alterations of CDR-H3 conformation. We counter that the conformational switch may not proceed through a full order-disorder transition, with a large entropic effect, but may switch between two relatively ordered states, with smaller entropic effects, much like a door swinging between completely open and completely shut. Also, the thermodynamics, performed on an isolated envelope peptide, have a surprisingly low entropic signature, considering that the peptide would be expected to go through an order-disorder transition during binding. What then might account for the different structural results, Fv versus Fab? Crystallization affects structure not only through solution conditions, but also through the requirement to pack molecules together in the crystal lattice. In the ligand-free Fab structure, the two Fabs in the asymmetric unit both make crystal contacts through CDR-H3. These contacts partially mimic interactions with HIV ligands (Fig. 1A to C). In other words, this structure may not represent a fully “ligand-free” state of 4E10, but, unluckily, recapitulates the ligand-bound state, resolving the potential contradiction. The CDR-H3 movement in our ligand-free Fv structure (3) also revealed a potential phosphate-binding site, explaining 4E10’s membrane-binding properties (3, 6, 10, 11). We found this result compelling, because we have argued that the ligand-bound state has no obvious feature accounting for phospholipid binding (3, 6). Rujas et al. (1) alternately interpret their results in terms of
Letter to the Editor
FUNDING INFORMATION HHS | National Institutes of Health (NIH) provided funding to Roland K Strong under grant number AI094419.
6.
REFERENCES
March 2016 Volume 90 Number 6
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8.
9.
10.
11.
Journal of Virology
jvi.asm.org
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Downloaded from http://jvi.asm.org/ on April 15, 2016 by TULANE UNIV
1. Rujas E, Gulzar N, Morante K, Tsumoto K, Scott JK, Nieva JL, Caaveiro JMM. 2015. Structural and thermodynamic basis of epitope binding by neutralizing and nonneutralizing forms of the anti-Hiv-1 antibody 4E10. J Virol 89:11975–11989. http://dx.doi.org/10.1128/JVI.01793-15. 2. Finton KA, Friend D, Jaffe J, Gewe M, Holmes MA, Larman HB, Stuart A, Larimore K, Greenberg PD, Elledge SJ, Stamatatos L, Strong RK. 2014. Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10. PLoS Pathog 10:e1004403. http://dx.doi.org/10.1371/journal.ppat.1004403. 3. Finton KA, Larimore K, Larman HB, Friend D, Correnti C, Rupert PB, Elledge SJ, Greenberg PD, Strong RK. 2013. Autoreactivity and exceptional CDR plasticity (but not unusual polyspecificity) hinder elicitation of the anti-HIV antibody 4E10. PLoS Pathog 9:e1003639. http://dx.doi .org/10.1371/journal.ppat.1003639. 4. Cardoso RM, Brunel FM, Ferguson S, Zwick M, Burton DR, Dawson PE, Wilson IA. 2007. Structural basis of enhanced binding of extended and helically constrained peptide epitopes of the broadly neutralizing HIV-1 antibody 4E10. J Mol Biol 365:1533–1544. http://dx.doi.org/10 .1016/j.jmb.2006.10.088. 5. Cardoso RM, Zwick MB, Stanfield RL, Kunert R, Binley JM, Katinger H, Burton DR, Wilson IA. 2005. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-
associated motif in gp41. Immunity 22:163–173. http://dx.doi.org/10 .1016/j.immuni.2004.12.011. Xu H, Song L, Kim M, Holmes MA, Kraft Z, Sellhorn G, Reinherz EL, Stamatatos L, Strong RK. 2010. Interactions between lipids and human anti-HIV antibody 4E10 can be reduced without ablating neutralizing activity. J Virol 84:1076 –1088. http://dx.doi.org/10.1128/JVI.02113-09. Correia BE, Ban YE, Holmes MA, Xu H, Ellingson K, Kraft Z, Carrico C, Boni E, Sather DN, Zenobia C, Burke KY, Bradley-Hewitt T, BruhnJohannsen JF, Kalyuzhniy O, Baker D, Strong RK, Stamatatos L, Schief WR. 2010. Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope. Structure 18:1116 –1126. http://dx.doi.org/10.1016/j.str.2010.06.010. Bird GH, Irimia A, Ofek G, Kwong PD, Wilson IA, Walensky LD. 2014. Stapled HIV-1 peptides recapitulate antigenic structures and engage broadly neutralizing antibodies. Nat Struct Mol Biol 21:1058 –1067. http: //dx.doi.org/10.1038/nsmb.2922. Rothlisberger D, Honegger A, Pluckthun A. 2005. Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J Mol Biol 347:773–789. http://dx.doi.org/10.1016/j.jmb.2005.01.053. Scherer EM, Leaman DP, Zwick MB, McMichael AJ, Burton DR. 2010. Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions. Proc Natl Acad Sci U S A 107:1529 –1534. http://dx.doi.org/10.1073/pnas.0909680107. Alam SM, Morelli M, Dennison SM, Liao HX, Zhang R, Xia SM, Rits-Volloch S, Sun L, Harrison SC, Haynes BF, Chen B. 2009. Role of HIV membrane in neutralization by two broadly neutralizing antibodies. Proc Natl Acad Sci U S A 106:20234 –20239. http://dx.doi.org/10.1073 /pnas.0908713106.
The Broadly Neutralizing, Anti-HIV Antibody 4E10: an Open and Shut Case? Roland K. Strong, Kathryn A. K. Finton Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
R
3274
jvi.asm.org
FIG 1 (A and B) Views of the two molecules in the asymmetric unit of the crystal structure of the “ligand-free” 4E10 Fab (PDB code 5CIP) (1), shown as cartoon ribbons colored to highlight different substructures. Chunks of neighboring 4E10 Fabs in the crystal lattice (yellow) are shown in a spherical representation. (C) The structure of the complex between the 4E10 Fab and an HIV envelope protein-derived peptide (2FX7) (5) is shown as in panels A and B. In these views, the packing constraints on the 4E10 CDR-H3 loop imposed by the epitope peptide ligand or neighboring Fabs are clearly revealed. (D) The phosphate-binding site in the 4E10 Fab/peptide complex is shown, with the molecular surface of the Fab, colored according to electrostatic potential, shown as a semitransparent overlay.
a phosphate-binding site observed in another 4E10 complex structure (8), where one of two key positively charged side chains bracketing the phosphate is a lysine in the bound peptide (Fig. 1D). However, since 4E10 membrane binding is a property of the ligand-free antibody (3, 6, 10, 11), this site is unlikely to explain this property.
Citation Strong RK, Finton KAK. 2016. The broadly neutralizing, anti-HIV antibody 4E10: an open and shut case? J Virol 90:3274 –3275. doi:10.1128/JVI.02935-15. Editor: F. Kirchhoff Address correspondence to Roland K. Strong, [email protected]. For the author reply, see doi:10.1128/JVI.02970-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Journal of Virology
March 2016 Volume 90 Number 6
Downloaded from http://jvi.asm.org/ on April 15, 2016 by TULANE UNIV
ujas et al. (1) recently studied the recognition mechanism of the anti-HIV antibody 4E10. We completely agree with their overall conclusion, that 4E10 “must recognize an antigenic structure more complex than just the linear ␣-helical epitope,” which echoes our own analysis (2): “4E10 may make contacts to elements of Env outside of the linear 4E10 epitope.” Their ligand-free Fab structure showed “that the CDR-H3 loop does not undergo significant conformational changes during recognition of the epitope,” consistent with thermodynamics that “ruled out major conformational changes of CDR-H3.” However, our structure of the ligand-free 4E10 Fv (3) had revealed a dramatic restructuring of CDR-H3, compared to Fv or Fab structures bound to HIV peptides (4–7). The difference was accounted for by the “low pH required to crystallize the Fv construct” and/or by “different structural stabilities” (1). Crystallization conditions can affect structure, and our ligand-free Fv crystallization conditions were nonphysiological, as noted by others (8). However, these conditions are not “extreme” in terms of protein crystallization overall. Solution thermostabilities of Fvs are also typically lower than those of Fabs (9), partly because Fvs lack the interchain disulfide that stabilizes Fabs. Since we had carefully shown that the 4E10 Fv retains the structure, binding, and neutralization properties of the 4E10 Fab (6), these explanations may not fully account for the apparent discrepancy. Rojas et al. state that the binding thermodynamics also argue against major alterations of CDR-H3 conformation. We counter that the conformational switch may not proceed through a full order-disorder transition, with a large entropic effect, but may switch between two relatively ordered states, with smaller entropic effects, much like a door swinging between completely open and completely shut. Also, the thermodynamics, performed on an isolated envelope peptide, have a surprisingly low entropic signature, considering that the peptide would be expected to go through an order-disorder transition during binding. What then might account for the different structural results, Fv versus Fab? Crystallization affects structure not only through solution conditions, but also through the requirement to pack molecules together in the crystal lattice. In the ligand-free Fab structure, the two Fabs in the asymmetric unit both make crystal contacts through CDR-H3. These contacts partially mimic interactions with HIV ligands (Fig. 1A to C). In other words, this structure may not represent a fully “ligand-free” state of 4E10, but, unluckily, recapitulates the ligand-bound state, resolving the potential contradiction. The CDR-H3 movement in our ligand-free Fv structure (3) also revealed a potential phosphate-binding site, explaining 4E10’s membrane-binding properties (3, 6, 10, 11). We found this result compelling, because we have argued that the ligand-bound state has no obvious feature accounting for phospholipid binding (3, 6). Rujas et al. (1) alternately interpret their results in terms of
Letter to the Editor
FUNDING INFORMATION HHS | National Institutes of Health (NIH) provided funding to Roland K Strong under grant number AI094419.
6.
REFERENCES
March 2016 Volume 90 Number 6
7.
8.
9.
10.
11.
Journal of Virology
jvi.asm.org
3275
Downloaded from http://jvi.asm.org/ on April 15, 2016 by TULANE UNIV
1. Rujas E, Gulzar N, Morante K, Tsumoto K, Scott JK, Nieva JL, Caaveiro JMM. 2015. Structural and thermodynamic basis of epitope binding by neutralizing and nonneutralizing forms of the anti-Hiv-1 antibody 4E10. J Virol 89:11975–11989. http://dx.doi.org/10.1128/JVI.01793-15. 2. Finton KA, Friend D, Jaffe J, Gewe M, Holmes MA, Larman HB, Stuart A, Larimore K, Greenberg PD, Elledge SJ, Stamatatos L, Strong RK. 2014. Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10. PLoS Pathog 10:e1004403. http://dx.doi.org/10.1371/journal.ppat.1004403. 3. Finton KA, Larimore K, Larman HB, Friend D, Correnti C, Rupert PB, Elledge SJ, Greenberg PD, Strong RK. 2013. Autoreactivity and exceptional CDR plasticity (but not unusual polyspecificity) hinder elicitation of the anti-HIV antibody 4E10. PLoS Pathog 9:e1003639. http://dx.doi .org/10.1371/journal.ppat.1003639. 4. Cardoso RM, Brunel FM, Ferguson S, Zwick M, Burton DR, Dawson PE, Wilson IA. 2007. Structural basis of enhanced binding of extended and helically constrained peptide epitopes of the broadly neutralizing HIV-1 antibody 4E10. J Mol Biol 365:1533–1544. http://dx.doi.org/10 .1016/j.jmb.2006.10.088. 5. Cardoso RM, Zwick MB, Stanfield RL, Kunert R, Binley JM, Katinger H, Burton DR, Wilson IA. 2005. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-
associated motif in gp41. Immunity 22:163–173. http://dx.doi.org/10 .1016/j.immuni.2004.12.011. Xu H, Song L, Kim M, Holmes MA, Kraft Z, Sellhorn G, Reinherz EL, Stamatatos L, Strong RK. 2010. Interactions between lipids and human anti-HIV antibody 4E10 can be reduced without ablating neutralizing activity. J Virol 84:1076 –1088. http://dx.doi.org/10.1128/JVI.02113-09. Correia BE, Ban YE, Holmes MA, Xu H, Ellingson K, Kraft Z, Carrico C, Boni E, Sather DN, Zenobia C, Burke KY, Bradley-Hewitt T, BruhnJohannsen JF, Kalyuzhniy O, Baker D, Strong RK, Stamatatos L, Schief WR. 2010. Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope. Structure 18:1116 –1126. http://dx.doi.org/10.1016/j.str.2010.06.010. Bird GH, Irimia A, Ofek G, Kwong PD, Wilson IA, Walensky LD. 2014. Stapled HIV-1 peptides recapitulate antigenic structures and engage broadly neutralizing antibodies. Nat Struct Mol Biol 21:1058 –1067. http: //dx.doi.org/10.1038/nsmb.2922. Rothlisberger D, Honegger A, Pluckthun A. 2005. Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J Mol Biol 347:773–789. http://dx.doi.org/10.1016/j.jmb.2005.01.053. Scherer EM, Leaman DP, Zwick MB, McMichael AJ, Burton DR. 2010. Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions. Proc Natl Acad Sci U S A 107:1529 –1534. http://dx.doi.org/10.1073/pnas.0909680107. Alam SM, Morelli M, Dennison SM, Liao HX, Zhang R, Xia SM, Rits-Volloch S, Sun L, Harrison SC, Haynes BF, Chen B. 2009. Role of HIV membrane in neutralization by two broadly neutralizing antibodies. Proc Natl Acad Sci U S A 106:20234 –20239. http://dx.doi.org/10.1073 /pnas.0908713106.
