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

Tong Li,Jose Casas-Finet,Shahid Uddin,Donald J Jacobs,Dennis R Livesay,Malgorzata B Tracka,Bert L De Groot

doi:10.1371/journal.pcbi.1004327

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.

Highlights

The variable region of an antibody is composed of a structurally conserved fold that contains six complementarity-determining regions (CDRs), known as hypervariable regions
High affinity antibodies are produced during the course of an immune response by successive mutations to germline gene-encoded antibodies
The numbers of mutations located in the Fab and CDRs with respect to the GL sequence are 12/6, 9/6 and 9/5 for FA, CA and esterase catalytic (EA), respectively, indicating that 50% or more of the mutations occur in CDRs

Summary

Introduction

The variable region of an antibody is composed of a structurally conserved fold that contains six complementarity-determining regions (CDRs), known as hypervariable regions. The GL antibodies undergo further cycles of somatic mutations for affinity maturation (AM) and specificity improvement as the immune response proceeds, which can produce an astronomical number of unique antibodies. A variety of biochemical and structural studies reveal that the same germline gene-encoded antibodies allow promiscuous binding to diverse antigens, and even the same antigens by quite different somatic mutations [3,4,5]. Conformational flexibility and the induced-fit models, are commonly invoked to explain the conformational changes of antibodies during affinity maturation. Conformational flexibility assumes GL antibodies retain a degree of structural plasticity in their backbone in order to bind a number of different unrelated antigens, a capacity referred to here as polyspecificity [3, 6]. Regardless of the explanation, it is clear that flexibility/rigidity is changed, which is closely related to the binding affinity and specificity of antigens [4, 5, 10,11,12,13]

Methods

Results

Conclusion