Combining genetic and biochemical approaches to identify functional molecular contact points

Abstract

Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins and protein complexes which are resistant to characterization by other methods.

Fig. 1. Summary of how differential exposure of mAb epitopes can lead to identification of protein-protein contacts. (A)

Under normal conditions only some of the antibodies can bind their epitopes due to occlusion of the epitopes by the ligand bound to the contact site. (B) Treatment with partially denaturing buffer dissociates the protein contact site, exposing the monoclonal antibody epitopes. Mutagenesis (C) and peptide competition (D) at the contact site can both disrupt binding of the ligand for the motif and expose the epitopes in a pattern similar to that induced by the partially denaturing buffer.

Fig. 2. Differential immunoprecipitation using high and low detergent buffers.

An example of differential immunoprecipitation in RIPA and IPP150 buffers. Unprecipitated in vitro translation sample and immunoprecipitation with a polyclonal antibody are controls. Note that mAbs 5, 6 and 10 immunoprecipitate P only under partially denaturing conditions. This figure was originally published in (9).

Fig. 3. Sequence homology aligment of multiple hepadnaviruses.

P sequences from eight hepadnaviruses were aligned. Sequences with a >80% homology are shaded green. The T3 motif is indicated. DHBV, Duck hepatitis B virus, SHBV, Stork hepatitis B virus, HHBV, Heron hepatitis B virus, RGHBV, Ross’ goose hepatitis B virus, WHV, Woodchuck hepatitis virus, GSHV, Ground squirrel hepatitis virus, WMHBV, Wooly monkey hepatitis B virus, HBV, Hepatitis B virus. Modified from a figure originally published in (9).

Fig. 4. Mutations can expose epitopes that are normally obscured.

P with a mutation that did not affect binding at the contact site (Y181F) or with mutations that did disrupt binding (I179D/L180D) were immunoprecipitated with the indicated antibodies in IPP150 buffer. The epitopes for mAbs 5, 6 and 10 were exposed both by mutations at the contact site and high detergent (compare Figs. 2 and 4). This figure was originally published in (9).

Fig. 5. Mutations can alter partial proteolysis pattern.

P was partially digested with papain and the fragments were resolved by SDS-PAGE. Lanes 1-4 show wild-type P and lanes 5-8 show P with mutations which disrupt the contact site on P. ε is a RNA stem loop required for proper folding and activation of P. Note the lack of the protected fragment in the digestion of mutant protein (compare lanes 4 and 8).

Fig. 6. Monoclonal antibodies can inhibit protein function.

In vitro translated P was incubated with the indicated monoclonal antibodies and a DNA priming assay was performed. Priming signals were normalized to activity of P without antibody. NM6 is an irrelevant control antibody. This figure was originally published in (9).

Fig. 7. Soluble peptides containing the putative binding site can competitively inhibit protein function.

P was translated in vitro in the presence of the T3 peptide or irrelevant MBP peptide. Priming was initiated by addition of MgCl₂ and [α-³²P]dGTP. Priming signal was normalized to the activity of P without DMSO or peptide. Peptide concentration is in mM and error bars are the standard deviation from 4 experiments. This figure was originally published in (9).