Application of antihelix antibodies in protein structure determination

Abstract

Antibodies are indispensable tools in protein engineering and structural biology. Antibodies suitable for structural studies should recognize the 3-dimensional (3D) conformations of target proteins. Generating such antibodies and characterizing their complexes with antigens take a significant amount of time and effort. Here, we show that we can expand the application of well-characterized antibodies by “transplanting” the epitopes that they recognize to proteins with completely different structures and sequences. Previously, several antibodies have been shown to recognize the alpha-helical conformation of antigenic peptides. We demonstrate that these antibodies can be made to bind to a variety of unrelated “off-target” proteins by modifying amino acids in the preexisting alpha helices of such proteins. Using X-ray crystallography, we determined the structures of the engineered protein–antibody complexes. All of the antibodies bound to the epitope-transplanted proteins, forming accurately predictable structures. Furthermore, we showed that binding of these antihelix antibodies to the engineered target proteins can modulate their catalytic activities by trapping them in selected functional states. Our method is simple and efficient, and it will have applications in protein X-ray crystallography, electron microscopy, and nanotechnology.

Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2

“…However, in many cases (including GPCRs), the production of such fusion proteins often leads to loss of activity or a failure to yield well diffracting crystals 45 . There are also many examples of using small molecule additives and auxiliary proteins, sometimes termed ‘crystallization chaperones’, including antibodies, nanobodies/FAB fragments, to aid crystallization 5 , 46 , 47 . Nanobodies have elicited interest in chaperoning protein crystallization due to their ability to reduce conformational heterogeneity and shield unproductive surfaces from solvents, whilst extending crystallographically productive surfaces to form crystal contacts 4 , 47 .…”

“…Nanobodies have elicited interest in chaperoning protein crystallization due to their ability to reduce conformational heterogeneity and shield unproductive surfaces from solvents, whilst extending crystallographically productive surfaces to form crystal contacts 4 , 47 . However, generating antibodies/nanobodies and characterizing their complexes with protein of interest is time-consuming and can lead to structures that are not biologically representative 46 , 47 .…”

Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

“…Even considering all their attributes, the use of Fabs for structural biology applications is still limited because of the requirement that they need to be generated to the particular target system being studied. Kim et al have proposed the use of anti-helix antibodies generated by "epitope transplant" to offtarget proteins as versatile chaperones in structural biology 31 . However, this technology has its own set of challenges 32 .…”

Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins

“…For instance, we have designed and implemented universal Fab based fiducials to a BRIL fusion (21,22), to different classes of trimeric G-proteins (23,24), and to portable stem-loop RNA motifs (25). Additionally, a variety of other types of portable motifs have been developed (26,27), some of which involve nanobodies applied as the portable motif in a different context (28).…”