Limits...
Key mutations stabilize antigen-binding conformation during affinity maturation of a broadly neutralizing influenza antibody lineage.

Xu H, Schmidt AG, O'Donnell T, Therkelsen MD, Kepler TB, Moody MA, Haynes BF, Liao HX, Harrison SC, Shaw DE - Proteins (2015)

Bottom Line: We have now used molecular dynamics simulations and existing crystal structures to identify potentially key maturation mutations, and we have characterized their effects on the CDR H3 loop and on antigen binding using further simulations and experimental affinity measurements, respectively.As few as two single-site mutations in each pathway can confer substantial loop stability, but none of them confers experimentally detectable stability on its own.Our results support models of the germinal center reaction in which two or more mutations can occur without concomitant selection and show how divergent pathways have yielded functionally equivalent antibodies.

View Article: PubMed Central - PubMed

Affiliation: D. E. Shaw Research, New York, New York, 10036.

Show MeSH

Related in: MedlinePlus

Log-log plot of association rate constants ka versus the fraction of antigen-binding conformation fC of various mutant Fabs. (A) All Fabs studied by MD simulations (Tables I and Supporting Information SI). The qualitative agreement between fC estimated from MD simulations and ka measured in kinetic experiments supports the finding that maturation in the CH65 lineage is driven by the stabilization of the CDR H3 loop in the binding conformation. The Pearson correlation coefficient between fC and ka, when both are available, is 0.26; perfect correlation is not to be expected, because factors other than CDR H3 loop stability also contribute to ka. Fabs whose affinities are below the interferometry sensitivity limit are represented by gray circles at an arbitrary vertical position, because their ka values are unknown. The mature antibodies are colored green. The hybrid Fab CH65HUCAL binds HA with intermediate affinity despite the low fC value estimated from MD simulations; its low fC value might be due to a lack of convergance of the MD simulations of finite length. (B) and (C) highlight the effects of mutations in CH65 (B) and CH67 (C) branches. The solid black arrows correspond to “forward” mutations (i.e., mutations that occurred in the maturation from the UCA to the mature antibody). The dashed arrows correspond to “reverse” mutations (i.e., mutations from the mature antibody to the UCA). The dotted black arrows correspond to the artificial mutation R104NH. Fabs whose affinities are below the interferometry sensitivity limit are shown in the gray box as bands at their respective fC values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4368477&req=5

fig03: Log-log plot of association rate constants ka versus the fraction of antigen-binding conformation fC of various mutant Fabs. (A) All Fabs studied by MD simulations (Tables I and Supporting Information SI). The qualitative agreement between fC estimated from MD simulations and ka measured in kinetic experiments supports the finding that maturation in the CH65 lineage is driven by the stabilization of the CDR H3 loop in the binding conformation. The Pearson correlation coefficient between fC and ka, when both are available, is 0.26; perfect correlation is not to be expected, because factors other than CDR H3 loop stability also contribute to ka. Fabs whose affinities are below the interferometry sensitivity limit are represented by gray circles at an arbitrary vertical position, because their ka values are unknown. The mature antibodies are colored green. The hybrid Fab CH65HUCAL binds HA with intermediate affinity despite the low fC value estimated from MD simulations; its low fC value might be due to a lack of convergance of the MD simulations of finite length. (B) and (C) highlight the effects of mutations in CH65 (B) and CH67 (C) branches. The solid black arrows correspond to “forward” mutations (i.e., mutations that occurred in the maturation from the UCA to the mature antibody). The dashed arrows correspond to “reverse” mutations (i.e., mutations from the mature antibody to the UCA). The dotted black arrows correspond to the artificial mutation R104NH. Fabs whose affinities are below the interferometry sensitivity limit are shown in the gray box as bands at their respective fC values.

Mentions: We examined the influence of the light-chain mutations just described when they are introduced into the last common intermediate, I-2. (The I-2 light chain has no somatic mutations and hence is equivalent to the UCA light chain.) To test whether the above mutations indeed establish contacts that help stabilize the CDR H3 loop, we performed simulations of the Fabs of mutants I-2HUCALD49Y (the heavy chain of I-2 and the light chain of the UCA with mutation D49Y), I-2HUCALY48C, I-2HUCALD49Y,Y48C, I-2HUCALH33D, I-2HUCALS31R, I-2HUCALH33D,S31R, and I-2. From these simulations, we computed fC, the fraction of the apo antibody in the antigen-binding conformation (i.e., the equilibrium population of that state), and the corresponding conformational equilibrium constant, KC = fC/(1 − fC), for each mutant Fab (Table1). The single forward mutation Y48LC in I-2 produced only a modest increase in KC, and D49LY had a deleterious effect (Fig. 3 and Table1). Only when the two were both present did KC in the CH65 branch increase substantially. We observed a similar effect in the CH67 branch: only when both forward mutations, H33LD and S31LR, appeared together did KC increase significantly. Both double mutants, I-2HUCALD49Y,Y48C and I-2HUCALH33D,S31R, stabilized the CDR H3 loop in the antigen-binding conformation such that the corresponding KC values were similar to those of their respective mature antibodies, CH65 and CH67. The large KC values estimated from the simulations for the two double mutants predict that their affinity for HA will be substantially higher than that of I-2.


Key mutations stabilize antigen-binding conformation during affinity maturation of a broadly neutralizing influenza antibody lineage.

Xu H, Schmidt AG, O'Donnell T, Therkelsen MD, Kepler TB, Moody MA, Haynes BF, Liao HX, Harrison SC, Shaw DE - Proteins (2015)

Log-log plot of association rate constants ka versus the fraction of antigen-binding conformation fC of various mutant Fabs. (A) All Fabs studied by MD simulations (Tables I and Supporting Information SI). The qualitative agreement between fC estimated from MD simulations and ka measured in kinetic experiments supports the finding that maturation in the CH65 lineage is driven by the stabilization of the CDR H3 loop in the binding conformation. The Pearson correlation coefficient between fC and ka, when both are available, is 0.26; perfect correlation is not to be expected, because factors other than CDR H3 loop stability also contribute to ka. Fabs whose affinities are below the interferometry sensitivity limit are represented by gray circles at an arbitrary vertical position, because their ka values are unknown. The mature antibodies are colored green. The hybrid Fab CH65HUCAL binds HA with intermediate affinity despite the low fC value estimated from MD simulations; its low fC value might be due to a lack of convergance of the MD simulations of finite length. (B) and (C) highlight the effects of mutations in CH65 (B) and CH67 (C) branches. The solid black arrows correspond to “forward” mutations (i.e., mutations that occurred in the maturation from the UCA to the mature antibody). The dashed arrows correspond to “reverse” mutations (i.e., mutations from the mature antibody to the UCA). The dotted black arrows correspond to the artificial mutation R104NH. Fabs whose affinities are below the interferometry sensitivity limit are shown in the gray box as bands at their respective fC values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368477&req=5

fig03: Log-log plot of association rate constants ka versus the fraction of antigen-binding conformation fC of various mutant Fabs. (A) All Fabs studied by MD simulations (Tables I and Supporting Information SI). The qualitative agreement between fC estimated from MD simulations and ka measured in kinetic experiments supports the finding that maturation in the CH65 lineage is driven by the stabilization of the CDR H3 loop in the binding conformation. The Pearson correlation coefficient between fC and ka, when both are available, is 0.26; perfect correlation is not to be expected, because factors other than CDR H3 loop stability also contribute to ka. Fabs whose affinities are below the interferometry sensitivity limit are represented by gray circles at an arbitrary vertical position, because their ka values are unknown. The mature antibodies are colored green. The hybrid Fab CH65HUCAL binds HA with intermediate affinity despite the low fC value estimated from MD simulations; its low fC value might be due to a lack of convergance of the MD simulations of finite length. (B) and (C) highlight the effects of mutations in CH65 (B) and CH67 (C) branches. The solid black arrows correspond to “forward” mutations (i.e., mutations that occurred in the maturation from the UCA to the mature antibody). The dashed arrows correspond to “reverse” mutations (i.e., mutations from the mature antibody to the UCA). The dotted black arrows correspond to the artificial mutation R104NH. Fabs whose affinities are below the interferometry sensitivity limit are shown in the gray box as bands at their respective fC values.
Mentions: We examined the influence of the light-chain mutations just described when they are introduced into the last common intermediate, I-2. (The I-2 light chain has no somatic mutations and hence is equivalent to the UCA light chain.) To test whether the above mutations indeed establish contacts that help stabilize the CDR H3 loop, we performed simulations of the Fabs of mutants I-2HUCALD49Y (the heavy chain of I-2 and the light chain of the UCA with mutation D49Y), I-2HUCALY48C, I-2HUCALD49Y,Y48C, I-2HUCALH33D, I-2HUCALS31R, I-2HUCALH33D,S31R, and I-2. From these simulations, we computed fC, the fraction of the apo antibody in the antigen-binding conformation (i.e., the equilibrium population of that state), and the corresponding conformational equilibrium constant, KC = fC/(1 − fC), for each mutant Fab (Table1). The single forward mutation Y48LC in I-2 produced only a modest increase in KC, and D49LY had a deleterious effect (Fig. 3 and Table1). Only when the two were both present did KC in the CH65 branch increase substantially. We observed a similar effect in the CH67 branch: only when both forward mutations, H33LD and S31LR, appeared together did KC increase significantly. Both double mutants, I-2HUCALD49Y,Y48C and I-2HUCALH33D,S31R, stabilized the CDR H3 loop in the antigen-binding conformation such that the corresponding KC values were similar to those of their respective mature antibodies, CH65 and CH67. The large KC values estimated from the simulations for the two double mutants predict that their affinity for HA will be substantially higher than that of I-2.

Bottom Line: We have now used molecular dynamics simulations and existing crystal structures to identify potentially key maturation mutations, and we have characterized their effects on the CDR H3 loop and on antigen binding using further simulations and experimental affinity measurements, respectively.As few as two single-site mutations in each pathway can confer substantial loop stability, but none of them confers experimentally detectable stability on its own.Our results support models of the germinal center reaction in which two or more mutations can occur without concomitant selection and show how divergent pathways have yielded functionally equivalent antibodies.

View Article: PubMed Central - PubMed

Affiliation: D. E. Shaw Research, New York, New York, 10036.

Show MeSH
Related in: MedlinePlus