Welcome to the Recombinant Antibody Network

@article{pmid41481455,

title = {E2 variants for probing E3 ubiquitin ligase activities},

author = {Jiale Du and Gisele A Andree and Daniel Horn-Ghetko and Luca Stier and Jaspal Singh and Sebastian Kostrhon and Leo Kiss and Matthias Mann and Sachdev S Sidhu and Brenda A Schulman},

doi = {10.1073/pnas.2524899122},

issn = {1091-6490},

year  = {2026},

date = {2026-01-01},

urldate = {2026-01-01},

journal = {Proc Natl Acad Sci U S A},

volume = {123},

number = {1},

pages = {e2524899122},

abstract = {E3 ligases partner with E2 enzymes to regulate vast eukaryotic biology. The hierarchical nature of these pairings, with >600 E3s and ~40 E2s in humans, necessitates that E2s cofunction with numerous different E3s. Here, focusing on E3s in the RING-between-RING (RBR) family and their partner UBE2L3 and UBE2D-family E2s, we report an approach to interrogate selected pathways. We screened phage-displayed libraries of structure-based E2 variants (E2Vs) to discover enzymes with enhanced affinity and specificity toward half of all RBR E3 ligases (ARIH1, ARIH2, ANKIB1, CUL9, HOIL1, HOIP, and RNF14). Collectively, these E2Vs allowed distinguishing actions of different cofunctioning E3s, obtaining high-resolution cryogenic Electron Microscopy (cryo-EM) structures of an RBR E3 in the context of a substrate-bound multiprotein complex, and profiling an endogenous RBR E3 response to an extracellular stimulus. Overall, we anticipate that E2V technology will be a generalizable tool to enable in-depth mechanistic and structural analysis of E3 ligase functions, and mapping their activity states and protein partners in cellular signaling cascades.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid41446272,

title = {Conformation-specific Antibody Deciphers K27-linked Ubiquitination in Chaperone-Mediated Proteostasis},

author = {Chengxiao Han and Yicheng Weng and Qingyun Zheng and Qian Qu and Satchal K Erramilli and Zhen Su and Yujuan Duan and Yunxi Han and Xiaoguo Zhai and Jingxian Li and Anthony A Kossiakoff and Man Pan and Minglei Zhao and Lei Liu and Yuanyuan Yu},

doi = {10.64898/2025.12.18.695067},

issn = {2692-8205},

year  = {2025},

date = {2025-12-01},

urldate = {2025-12-01},

journal = {bioRxiv},

abstract = {Lysine 27 (K27)-linked polyubiquitination plays critical yet incompletely defined roles in proteostasis, innate immunity, and disease progression; however, investigations into this process have long been hindered by its extremely low abundance and the lack of conformation-specific enrichment tools. Herein, we describe the development of a long-sought conformation-specific antibody, K27-IgG, which can selectively recognize-among all ubiquitin chain types-the unique architecture of K27-linked polyubiquitin (K27-polyUb) characterized by a distinct buried K27-isopeptide bond, with high affinity (KD = 4.66 nM). This antibody was derived from synthetic antibodies initially generated via phage display, using chemically synthesized K27-linked diubiquitin (K27-diUb) as the antigen. High-resolution co-crystal structures uncovered the unique K27-diUb interface targeted by these sAbs. Subsequent reformatting of these sAbs into a full-length human immunoglobulin G (IgG) scaffold yielded K27-IgG, notably exhibiting markedly enhanced affinity without compromising selectivity. Using K27-IgG as a tool, we achieved sensitive detection and immunoprecipitation (IP) of endogenous K27-polyUb in cells, and delineated the intracellular interaction landscape of K27-polyUb through complementary proteomic approaches. Two key findings emerged: 1) The molecular chaperone DNAJB1 is a specific reader of K27-linked ubiquitin chains (but not other linkages) and that K27-polyUb chains themselves exhibit chaperone-like activity, suggesting a novel mechanism by which K27-polyUb regulates chaperone-mediated proteostasis; 2) The E2 enzyme UBE2Q1 assembles K27-diUb, identifying it as a potential writer for this ubiquitin chain topology. Collectively, this study establishes K27-IgG as a robust tool for deciphering the K27-linked ubiquitin code, thereby opening new avenues for investigating the biological functions of K27-linked polyubiquitination.nnHIGHLIGHTS: First K27-linkage conformation-specific antibody with nanomolar affinity overcomes a major barrier in the field.K27-IgG unlocks functional mapping of the K27 ubiquitin landscape under proteotoxic stress.Molecular chaperone DNAJB1 is a selective "reader" of K27-linked ubiquitin chains.K27 chains possess intrinsic chaperone activity, enabling protein refolding and suppressing aggregation.E2 enzyme UBE2Q1 is a "writer" that directly assembles K27-linked ubiquitin chains.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid41298376,

title = {Mechanistic basis of antimicrobial resistance mediated by the phosphoethanolamine transferase MCR-1},

author = {Allen P Zinkle and Mariana Bunoro Batista and Carmen M Herrera and Satchal K Erramilli and Brian Kloss and Khuram U Ashraf and Kamil Nosol and Guozhi Zhang and Rosemary J Cater and Michael T Marty and Anthony A Kossiakoff and M Stephen Trent and Rie Nygaard and Phillip J Stansfeld and Filippo Mancia},

doi = {10.1038/s41467-025-65515-3},

issn = {2041-1723},

year  = {2025},

date = {2025-11-01},

urldate = {2025-11-01},

journal = {Nat Commun},

volume = {16},

number = {1},

pages = {10516},

abstract = {Polymyxins are used to treat infections caused by multidrug-resistant Gram-negative bacteria. They are cationic peptides that target the negatively charged lipid A component of lipopolysaccharides, disrupting the outer membrane and lysing the cell. Polymyxin resistance is conferred by inner-membrane enzymes, such as phosphoethanolamine transferases, which add positively charged phosphoethanolamine to lipid A. Here, we present the structure of MCR-1, a plasmid-encoded phosphoethanolamine transferase, in its liganded form. The phosphatidylethanolamine donor substrate is bound near the active site in the periplasmic domain, and lipid A is bound over 20 Å away, within the transmembrane region. Integrating structural, biochemical, and drug-resistance data with computational analyses, we propose a two-state model in which the periplasmic domain rotates to bring the active site to lipid A, near the preferential phosphate modification site for MCR-1. This enzymatic mechanism may be generally applicable to other phosphoform transferases with large, globular soluble domains.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}