Using phage display for rational engineering of a higher affinity humanized 3’phosphohistidine-specific antibody
Bibliographic record
Abstract
Abstract Histidine phosphorylation (pHis) is a non-canonical post-translational modification (PTM) that is historically understudied due to a lack of robust reagents that are required for its investigation, such as high affinity pHis-specific antibodies. Engineering pHis-specific antibodies is very challenging due to the labile nature of the phosphoramidate (P-N) bond and the stringent requirements for selective recognition of the two isoforms, 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis). Here, we present a strategy for in vitro engineering of antibodies for detection of native 3-pHis targets. Specifically, we humanized the rabbit SC44-8 anti-3-pTza (a stable 3-pHis mimetic) mAb into a scaffold (herein referred to as hSC44) that was suitable for phage display. We then constructed six unique Fab phage-displayed libraries using the hSC44 scaffold and selected high affinity 3-pHis binders. Our selection strategy was carefully designed to enrich antibodies that bound 3-pHis with high affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32F L , the best engineered antibody, has an ∼10-fold higher affinity for 3-pHis than the parental hSC44. Eleven new Fab structures, including the first reported antibody-pHis peptide structures were solved by X-ray crystallography. Structural and quantum mechanical calculations provided molecular insights into 3-pHis and 3-pTza discrimination by different hSC44 variants and their affinity increase obtained through in vitro engineering. Furthermore, we demonstrate the utility of these newly developed high-affinity 3-pHis-specific antibodies for recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Overall, our work describes a general method for engineering PTM-specific antibodies and provides a set of novel antibodies for further investigations of the role of 3-pHis in cell biology. Significance Statement Histidine phosphorylation is an elusive PTM whose role in mammalian cell biology is largely unknown due to the lack of robust tools and methods for its analysis. Here we report the development of antibodies with unprecedented affinity and specificity towards 3-pHis and present the first crystal structures of a pHis peptide in complex with an antibody. Finally, we show how these antibodies can be used in standard molecular biology workflows to investigate pHis-dependent biology.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".