Abstract
Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope.
Highlights
We found that deletion of the tip of the CDR H3 (YDFWSGYPP) resulted in a variant of 10E8v4 that eluted as a single size-exclusion chromatography (SEC) peak
These results indicated the anomalous peak profiles and retained positions of peaks on SEC to be a result of interactions of the hydrophobic CDR H3 of show the disulfide bond peak to run anomalously longer on SEC, when compared to antibody VRC01, a well characterized HIV-1 neutralizing antibody that behaves as an expected monoclonal antibody by SEC [27] (Figure 1A,B)
These results indicated the anomalous peak profiles and retained positions of peaks on SEC to be a result of interactions of the hydrophobic CDR H3 of 10E8v4 with thewith separation matrix, while stabilization of the active conformation of the CDR
Summary
Antibody 10E8 is one of the broadest monoclonal antibodies far identified that effectively neutralizes diverse strains of HIV-1 [9,10,11] It targets the membrane-proximal external region (MPER) of the HIV-1 viral spike with a highly hydrophobic antigen-binding region [12]. To investigate the generality of cis-proline isomerization with other MPER-directed antibodies, we introduced Tyr-Pro or Pro-Pro motif to the CDR H3s of 4E10 and VRC42, and performed SEC Both of these antibodies have a single proline in their CDR H3s, and, this proline assumes a trans-conformation [21,40], both of these antibodies showed slightly extended interactions on SEC and evidence of multi-peak behavior (Figure 6). These results indicate witharomatic aromaticside sidechains chains beforethe theproline prolinecause causetheir their anomalous anomalous SEC behavior. before behavior.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
