Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

Abstract

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

“…We postulated that the energy compensation at the Fabs of the IgAs variants to accommodate the flexibility changes upon balance at the Her2-binding site (Table 1 and 3) to maintain the Her2-binding ability. As a result, our findings partially agree with several previous studies [40][41][42] that the antigen-binding region requires a certain level of rigidity. On the contrary, other studies [43,44] suggested no significant conformational differences found in these regions, perhaps due to the different antigens.…”

Mutations in Antibody Fragments Modulate Allosteric Response Via Hydrogen-Bond Network Fluctuations

“…As discussed above, the MPM correctly identifies the phosphorylation site as allosteric in three chemotaxis protein Y orthologs (26), which is the only conserved site identified across the set. As with the MPM results, our collective results reveal CC to be very sensitive to sequence and structural variations across protein families (28,30,31,(44)(45)(46)(47)(48)(49)(50), which is in complete agreement with various experimental comparisons of allosteric effects across protein families. Going as far back as 1970 (51), quantifiable differences in allosteric response have been used as a taxonomic marker in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase family.…”

“…Our prior works have extensively evaluated how sequence variations lead to redistributions within rigidity and flexibility in antibody fragments (31,44,45). Interestingly, thermostabilizing mutations often locally rigidify the protein due to local optimization of the HBN, while the entropic cost of this rigidification is offset by a corresponding increase in flexibility in regions far removed from the mutation.…”

Antigen binding allosterically promotes Fc receptor recognition

“…[5][6][7] Recent studies suggested allosteric effects during antibody-antigen recognition 8 , with both the variable and constant domains playing a role. [9][10][11][12][13][14] For instance, our recent work on crenezumab suggested that antibodies with identical variable domains, but different constant domains, have significantly different affinities to amyloid beta (Aβ). 15 Engineering CH and CL in trastuzumab and pertuzumab recombinant models also affect antigen-binding.…”