Labeled and linked: The small-molecule binding site of Escherichia coli lipoic acid ligase was re-engineered to accept a fluorinated aryl azide probe in place of lipoic acid. Labeling with this mutant is highly specific for LAP fusion proteins. In cell lysate, FK506 binding protein was labeled and rapamycin-dependent photo-cross-linking to its interaction partner was demonstrated. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z802088_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
The focus of the antibody-drug conjugate (ADC) field is shifting toward development of site-specific, next-generation ADCs to address the issue of heterogeneity, metabolic instability, conjugatability, and less than ideal therapeutic index associated with the conventional (heterogeneous) ADCs. It is evident from the recent literature that the site of conjugation, the structure of the linker, and the physicochemical properties of the linker-payload all have a significant impact on the safety and efficacy of the resulting ADCs. Screening multiple linker-payloads on multiple sites of an antibody presents a combinatorial problem that necessitates high-throughput conjugation and purification methodology to identify ADCs with the best combination of site and payload. Toward this end, we developed a protein A/L-based solid-phase, site-specific conjugation and purification method that can be used to generate site-specific ADCs in a 96-well plate format. This solid-phase method has been shown to be versatile because of its compatibility with various conjugation functional handles such as maleimides, haloacetamides, copper free click substrates, and transglutaminase substrates. The application of this methodology was further expanded to generate dual labeled, site-specific antibody and Fab conjugates.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Escherichia coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine side chains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides (Nat. Biotechnol. 2007, 25, 1483−1487) and photo-cross-linkers (Angew. Chem., Int. Ed. 2008, 47, 7018−7021) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA's utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA's natural protein substrates have a conserved β-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called "LplA Acceptor Peptide" (LAP). A ∼107 library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a "LAP2" sequence with a kcat/Km of 0.99 μM−1 min−1, >70-fold better than our previous rationally designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483−1487), and only 8-fold worse than the kcat/Km values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.
The cover picture shows the use of site‐specific incorporation of N 2 ‐benzylguanosine into RNA as a method for controlling the binding sites occupied by proteins that contain double‐stranded RNA binding motifs (dsRBMs). A fragment of the Epstein–Barr virus‐encoded RNA 1 (EBER1) binds dsRBM I from the RNA‐dependent protein kinase (PKR) at two sites. Based on data from affinity cleavage experiments and molecular modeling, nucleotide positions were chosen in this RNA for incorporation of N 2 ‐benzyl guanosine, which places a bulky benzyl group in the minor groove of the RNA duplex. These site‐specific modifications lead to controlled inhibition of binding by PKR at the two different sites. This simple but effective technique can be further used to study similar dsRBM–RNA complexes. Modifications of duplex RNA like this that prevent dsRBMs from binding may be necessary in the development of RNA interference reagents (siRNAs) that will not interact with PKR or other double‐stranded RNA binding proteins. For more details see the article by P. A. Beal et al. on p. 383 ff.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)