Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.

A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.

DNA damage alters binding conformations of E. coli single-stranded DNA-binding protein.

Single-stranded DNA-binding proteins (SSBs) are essential cellular components, binding to transiently exposed regions of single-stranded DNA (ssDNA) with high affinity and sequence non-specificity to coordinate DNA repair and replication. Escherichia coli SSB (EcSSB) is a homotetramer that wraps variable lengths of ssDNA in multiple conformations (typically occupying either 65 or 35 nt), which is well studied across experimental conditions of substrate length, salt, pH, temperature, etc. In this work, we use atomic force microscopy to investigate the binding of SSB to individual ssDNA molecules. We introduce non-canonical DNA bases that mimic naturally occurring DNA damage, synthetic abasic sites, as well as a non-DNA linker into our experimental constructs at sites predicted to interact with EcSSB. By measuring the fraction of DNA molecules with EcSSB bound as well as the volume of protein bound per DNA molecule, we determine the protein binding affinity, cooperativity, and conformation. We find that, with only one damaged nucleotide, the binding of EcSSB is unchanged relative to its binding to undamaged DNA. In the presence of either two tandem abasic sites or a non-DNA spacer, however, the binding affinity associated with a single EcSSB tetramer occupying the full substrate in the 65-nt mode is greatly reduced. In contrast, the binding of two EcSSB tetramers, each in the 35-nt mode, is preserved. Changes in the binding and cooperative behaviors of EcSSB across these constructs can inform how genomic repair and replication processes may change as environmental damage accumulates in DNA.

Multiprotein E. coli SSB-ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions.

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB-ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.