Specific fluorine labeling of the HyHEL10 antibody affects antigen binding and dynamics.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

Association energetics of cross-reactive and specific antibodies.

HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.

Understanding antibody-antigen associations by molecular dynamics simulations: detection of important intra- and inter-molecular salt bridges.

1 NSec molecular dynamics (MD) simulation of anti-hen egg white antibody, HyHEL63 (HH63), complexed with HEL reveals important molecular interactions, not revealed in its X-ray crystal structure. These molecular interactions were predicted to be critical for the complex formation, based on structure-function studies of this complex and 3-other anti-HEL antibodies, HH8, HH10 and HH26, HEL complexes. All four antibodies belong to the same structural family, referred to here as HH10 family. Ala scanning results show that they recognize 'coincident epitopes'. 1 NSec explicit, with periodic boundary condition, MD simulation of HH63- HEL reveals the presence of functionally important saltbridges. Around 200 ps in vacuo and an additional 20 ps explicit simulation agree with the observations from 1 Nsec simulation. Intra-molecular salt-bridges predicted to play significant roles in the complex formation, were revealed during MD simulation. A very stabilizing saltbridge network, and another intra-molecular salt-bridge, at the binding site of HEL, revealed during the MD simulation, is proposed to predipose binding site geometry for specific binding. All the revealed saltbridges are present in one or more of the other three complexes and/or involve \"hot-spot\" epitope and paratope residues. Most of these charged epitope residues make large contribution to the binding free energy. The "hot spot" epitope residue Lys97Y, which significantly contributes to the free energy of binding in all the complexes, forms an intermolecular salt-bridge in several MD conformers. Our earlier computations have shown that this inter-molecular salt-bridge plays a significant role in determining specificity and flexibility of binding in the HH8-HEL and HH26-HEL complexes. Using a robust criterion of salt-bridge detection, this intermolecular salt-bridge was detected in the native structures of the HH8-HEL and HH26-HEL complexes, but was not revealed in the crystal structure of HH63-HEL complex. The electrostatic strength of this revealed saltbridge was very strong. During 1 Nsec MD simulation this salt-bridge networks with another inter-molecular salt-bridge to form an inter-molecular salt-bridge triad. Participation of Lys97Y in the formation of inter-molecular triad further validates the functional importance of Lys97Y in HH63-HEL associations. These results demonstrate that many important structural details of biomolecular interactions can be better understood when studied in a dynamic environment, and that MD simulations can complement and expand information obtained from static X-ray structure. This study also highlights "hot-spot" molecular interactions in HyHEL63-HEL complex.

Schweinfurthin A selectively inhibits proliferation and Rho signaling in glioma and neurofibromatosis type 1 tumor cells in a NF1-GRD-dependent manner.

Neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the nervous system. Patients typically develop many tumors over their lifetime, leading to increased morbidity and mortality. The NF1 gene, mutated in NF1, is also commonly mutated in sporadic glioblastoma multiforme (GBM). Because both NF1 and GBM are currently incurable, new therapeutic approaches are clearly needed. Natural products represent an opportunity to develop new therapies, as they have been evolutionarily selected to play targeted roles in organisms. Schweinfurthin A is a prenylated stilbene natural product that has previously shown specific inhibitory activity against brain and hematopoietic tumor lines. We show that patient-derived GBM and NF1 malignant peripheral nerve sheath tumor (MPNST) lines, as well as tumor lines derived from the Nf1-/+;Trp53-/+ (NPcis) mouse model of astrocytoma and MPNST are highly sensitive to inhibition by schweinfurthin A and its synthetic analogs. In contrast, primary mouse astrocytes are resistant to the growth inhibitory effects of schweinfurthin A, suggesting that schweinfurthin A may act specifically on tumor cells. Stable transfection of the GTPase-activating protein related domain of Nf1 into Nf1-/-;Trp53-/- astrocytoma cells confers resistance to schweinfurthin A. In addition, the profound effect of schweinfurthin A on dynamic reorganization of the actin cytoskeleton led us to discover that schweinfurthin A inhibits growth factor-stimulated Rho signaling. In summary, we have identified a class of small molecules that specifically inhibit growth of cells from both central and peripheral nervous system tumors and seem to act on NF1-deficient cells through cytoskeletal reorganization correlating to changes in Rho signaling.

Light chain somatic mutations change thermodynamics of binding and water coordination in the HyHEL-10 family of antibodies.

Thermodynamic and structural studies addressed the increased affinity due to L-chain somatic mutations in the HyHEL-10 family of affinity matured IgG antibodies, using ITC, SPR with van't Hoff analysis, and X-ray crystallography. When compared to the parental antibody H26L26, the H26L10 and H26L8 chimeras binding to lysozyme showed an increase in favorable DeltaG(o) of -1.2+/-0.1 kcal mol(-1) and -1.3+/-0.1 kcal mol(-1), respectively. Increase in affinity of the H26L10 chimera was due to a net increase in favorable enthalpy change with little difference in change in entropy compared to H26L26. The H26L8 chimera exhibited the greatest increase in favorable enthalpy but also showed an increase in unfavorable entropy change, with the result being that the affinities of both chimeras were essentially equivalent. Site-directed L-chain mutants identified the shared somatic mutation S30G as the dominant contributor to increasing affinity to lysozyme. This mutation was not influenced by H-chain somatic mutations. Residue 30L is at the periphery of the binding interface and S30G effects an increase in hydrophobicity and decrease in H-bonding ability and size, but does not make any new energetically important antigen contacts. A new 1.2-A structure of the H10L10-HEL complex showed changes in the pattern of both inter- and intra-molecular water bridging with no other significant structural alterations near the binding interface compared to the H26L26-HEL complex. These results highlight the necessity for investigating both the structure and the thermodynamics associated with introduced mutations, in order to better assess and understand their impact on binding. Furthermore, it provides an important example of how backbone flexibility and water-bridging may favorably influence the thermodynamics of an antibody-antigen interaction.

Differences in electrostatic properties at antibody-antigen binding sites: implications for specificity and cross-reactivity.

Antibodies HyHEL8, HyHEL10, and HyHEL26 (HH8, HH10, and HH26, respectively) recognize highly overlapping epitopes on hen egg-white lysozyme (HEL) with similar affinities, but with different specificities. HH8 binding to HEL is least sensitive toward mutations in the epitope and thus is most cross-reactive, HH26 is most sensitive, whereas the sensitivity of HH10 lies in between HH8 and HH26. Here we have investigated intra- and intermolecular interactions in three antibody-protein complexes: theoretical models of HH8-HEL and HH26-HEL complexes, and the x-ray crystal structure of HH10-HEL complex. Our results show that HH8-HEL has the lowest number and HH26-HEL has the highest number of intra- and intermolecular hydrogen bonds. The number of salt bridges is lowest in HH8-HEL and highest in HH26-HEL. The binding site salt bridges in HH8-HEL are not networked, and are weak, whereas, in HH26-HEL, an intramolecular salt-bridge triad at the binding site is networked to an intermolecular triad to form a pentad. The pentad and each salt bridge of this pentad are exceptionally stabilizing. The number of binding-site salt bridges and their strengths are intermediate in HH10-HEL, with an intramolecular triad. Our further calculations show that the electrostatic component contributes the most to binding energy of HH26-HEL, whereas the hydrophobic component contributes the most in the case of HH8-HEL. A "hot-spot" epitope residue Lys-97 forms an intermolecular salt bridge in HH8-HEL, and participates in the intermolecular pentad in the HH26-HEL complex. Mutant modeling and surface plasmon resonance (SPR) studies show that this hot-spot epitope residue contributes significantly more to the binding than an adjacent epitope residue, Lys-96, which does not form a salt bridge in any of the three HH-HEL complexes. Furthermore, the effect of mutating Lys-97 is most severe in HH26-HEL. Lys-96, being a charged residue, also contributes the most in HH26-HEL among the three complexes. The SPR results on these mutants also highlight that the apparent "electrostatic steering" on net on rates actually act at post-collision level stabilization of the complex. The significance of this work is the observed variations in electrostatic interactions among the three complexes. Our work demonstrates that higher electrostatics, both as a number of short-range electrostatic interactions and their contributions, leads to higher binding specificity. Strong salt bridges, their networking, and electrostatically driven binding, limit flexibilities through geometric constrains. In contrast, hydrophobic driven binding and low levels of electrostatic interactions are associated with conformational flexibility and cross-reactivity.

Temperature differentially affects encounter and docking thermodynamics of antibody--antigen association.

Using BIACORE SPR, we have examined the mechanism of temperature effects on the binding kinetics of two closely related antibody Fabs (H10 and H26) which recognize coincident epitopes on hen egg-white lysozyme (HEL), and whose association and dissociation kinetics are best described by the two-step conformational change model which we interpret as molecular encounter and docking. Time-course series data obtained at a series of six temperatures (6, 10, 15, 25, 30 and 37 degrees C) showed that temperature differentially affects the rate constants of the encounter and docking steps. Docking is more temperature-sensitive than the encounter step, and energetically less favorable at higher temperatures. At elevated temperatures, the time required for docking is longer and the apparent increase in off-rate reflects the greater proportion of the molecules failing to dock and remaining in the less stable encounter state. As a consequence, distribution of free energy change between the encounter and docking steps is altered. At physiological temperature (37 degrees C) the docking step of the H26 complex is energetically unfavorable and most complexes essentially do not dock. There is a significant decrease in total free energy change of the H26 complex at higher temperatures. Elevated temperature changes the rate-limiting step of H26--HEL association from the encounter to the docking step, but not that of H10--HEL. Our results indicate that the mechanism by which elevated temperature reduces the affinities of antigen--antibody complexes is to decrease the net docking rate, and/or stability of the docked complex; at higher temperatures, a smaller proportion of the complexes actually anneal to a more stable docked state. This mechanism may have broad applicability to other receptor--ligand complexes.