8UHT

anti-Phosphohistidine Fab hSC44.ck.20.elbow bound to Glu H1

Summary for 8UHT

• Entry DOI: 10.2210/pdb8uht/pdb
• Descriptor: hSC44.ck.20.elbow Fab heavy chain, hSC44.ck.20.elbow Fab light chain (3 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: 2
• Total formula weight: 47546.00

Authors

Kalagiri, R.,Stanfield, R.L.,Hunter, T.,Wilson, I.A. (deposition date: 2023-10-09, release date: 2025-03-19, Last modification date: 2026-07-15)

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.
Commun Chem, 8:381-381, 2025

PubMed Abstract: Histidine phosphorylation is a non-canonical post-translational modification (PTM), with 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis) isoforms, that is understudied due to a lack of robust reagents, including high-affinity pHis-specific antibodies. Engineering pHis antibodies is challenging due to the labile nature of its phosphoramidate (P-N) bond. We developed a strategy for in vitro engineering of antibodies for the detection of native 3-pHis targets, in which the rabbit SC44-8 anti-3-pTza mAb is humanized into a scaffold (hSC44) that is suitable for phage display. Six unique Fab phage-displayed hSC44 scaffold libraries were screened for antibodies that bound 3-pHis with higher affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32F, the best engineered antibody, has ~10-fold higher affinity for 3-pHis than parental hSC44. Eleven new Fab structures, including the first antibody-pHis peptide structures, together with structural and quantum mechanical calculations, provided molecular insights into 3-pHis and 3-pTza discrimination by hSC44.20N32F and the increased affinity obtained through engineering. We demonstrated the utility of these high-affinity 3-pHis-specific antibodies for the recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Our work describes a general method for engineering labile PTM-specific antibodies and provides novel antibodies for investigating the role of 3-pHis in cell biology.

PubMed: 41310398
DOI: 10.1038/s42004-025-01768-9

Experimental method

X-RAY DIFFRACTION (2.45 Å)