Co-Crystallization with Diabodies: A Universal Method for the Introduction of Synthetic Symmetry

Abstract

This work presents a method for introducing synthetic symmetry into protein crystallization samples using an antibody fragment termed a diabody (Dab). These Dabs contain two target binding sites and engineered disulfide bonds have been included to modulate Dab flexibility. The introduction of a disulfide bond promotes a preferred conformation and an increased likelihood of crystallization as observed in solution by small-angle X-ray scattering and in high-resolution crystal structures. To explore the capacity of engineered Dabs as crystallization chaperones, complexes between the engineered Dabs and HIV-1 reverse transcriptase (RT) bound to a high-affinity DNA aptamer were generated. This strategy increased the crystallization hit frequency obtained for RT-aptamer and the structure of a Dab-RT-DNA complex was determined to 3.0 A resolution. Introduction of synthetic symmetry using a Dab is a broadly applicable strategy, especially when monoclonal antibodies for a target have previously been identified.