8UIG

anti-Phosphohistidine Fab hSC44.ck.20 with 3pHis peptide

Summary for 8UIG

• Entry DOI: 10.2210/pdb8uig/pdb
• Descriptor: hSC44.ck.20 Fab heavy chain, hSC44.ck.20 Fab light chain, 3pHis peptide, ... (4 entities in total)
• Functional Keywords: anti-phosphohistidine antibody post-translational modification antibody humanization, immune system
• Biological source: Oryctolagus cuniculus; More
• Total number of polymer chains: 3
• Total formula weight: 48224.50

Authors

Kalagiri, R.,Stanfield, R.L.,Hunter, T.,Wilson, I.A. (deposition date: 2023-10-10, release date: 2025-03-19)

Primary citation

Martyn, G.D.,Kalagiri, R.,Veggiani, G.,Stanfield, R.L.,Choudhuri, I.,Sala, M.,Meisenhelder, J.,Chen, C.,Biswas, A.,Levy, R.M.,Lyumkis, D.,Wilson, I.A.,Hunter, T.,Sidhu, S.S.
Using phage display for rational engineering of a higher affinity humanized 3' phosphohistidine-specific antibody.
Biorxiv, 2024

PubMed 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 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, 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 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.

PubMed: 39574610
DOI: 10.1101/2024.11.04.621849

Experimental method

X-RAY DIFFRACTION (1.9 Å)