Development of a conformation-specific antibody (K27-IgG) with nanomolar affinity enables detection and functional characterization of K27-linked polyubiquitination, revealing that K27 chains possess intrinsic chaperone-like activity, are specifically recognized by the molecular chaperone DNAJB1 to regulate proteostasis, and are assembled by the E2 enzyme UBE2Q1, thereby establishing a complete writer-reader framework for this atypical ubiquitin modification.

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.

Highlights

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.