Interleukin 10 (IL-10) is a dimeric cytokine that plays a central role in suppressing inflammatory responses. These activities are dependent on the interaction of IL-10 with its high-affinity receptor (IL-10R1). This intermediate complex must subsequently recruit the low-affinity IL-10R2 chain before cell signaling can occur. Here we report the 2.9 A crystal structure of IL-10 bound to a soluble form of IL-10R1 (sIL-10R1). The complex consists of two IL-10s and four sIL-10R1 molecules. Several residues in the IL-10/sIL-10R1 interface are conserved in all IL-10 homologs and their receptors. The data suggests that formation of the active IL-10 signaling complex occurs by a novel molecular recognition paradigm where IL-10R1 and IL-10R2 both recognize the same binding site on IL-10.

Combinatorial libraries of nonbiological polymers and drug-like peptides could in principle be synthesized from unnatural amino acids by exploiting the broad substrate specificity of the ribosome. The ribosomal synthesis of such libraries would allow rare functional molecules to be identified using technologies developed for the in vitro selection of peptides and proteins. Here, we use a reconstituted E. coli translation system to simultaneously reassign 35 of the 61 sense codons to 12 unnatural amino acid analogues. This reprogrammed genetic code was used to direct the synthesis of a single peptide containing 10 different unnatural amino acids. This system is compatible with mRNA-display, enabling the synthesis of unnatural peptide libraries of 10 14 unique members for the in vitro selection of functional unnatural molecules. We also show that the chemical space sampled by these libraries can be expanded using mutant aminoacyltRNA synthetases for the incorporation of additional unnatural amino acids or by the specific posttranslational chemical derivitization of reactive groups with small molecules. This system represents a first step toward a platform for the synthesis by enzymatic tRNA aminoacylation and ribosomal translation of cyclic peptides comprised of unnatural amino acids that are similar to the nonribosomal peptides.

The biochemical flexibility of the cellular translation apparatus offers, in principle, a simple route to the synthesis of drug-like modified peptides and novel biopolymers. However, only Ϸ75 unnatural building blocks are known to be fully compatible with enzymatic tRNA acylation and subsequent ribosomal synthesis of modified peptides. Although the translation system can reject substrate analogs at several steps along the pathway to peptide synthesis, much of the specificity resides at the level of the aminoacyl-tRNA synthetase (AARS) enzymes that are responsible for charging tRNAs with amino acids. We have developed an AARS assay based on mass spectrometry that can be used to rapidly identify unnatural monomers that can be enzymatically charged onto tRNA. By using this assay, we have found 59 previously unknown AARS substrates. These include numerous side-chain analogs with useful functional properties. Remarkably, many ␤-amino acids, N-methyl amino acids, and ␣,␣-disubstituted amino acids are also AARS substrates. These previously unidentified AARS substrates will be useful in studies of the specificity of subsequent steps in translation and may significantly expand the number of analogs that can be used for the ribosomal synthesis of modified peptides.aminoacyl-tRNA synthetases ͉ enzyme specificity ͉ MALDI-TOF mass spectrometry ͉ translation

BackgroundThe application of in vitro translation to the synthesis of unnatural peptides may allow the production of extremely large libraries of highly modified peptides, which are a potential source of lead compounds in the search for new pharmaceutical agents. The specificity of the translation apparatus, however, limits the diversity of unnatural amino acids that can be incorporated into peptides by ribosomal translation. We have previously shown that over 90 unnatural amino acids can be enzymatically loaded onto tRNA.Methodology/Principal FindingsWe have now used a competition assay to assess the efficiency of tRNA-aminoacylation of these analogs. We have also used a series of peptide translation assays to measure the efficiency with which these analogs are incorporated into peptides. The translation apparatus tolerates most side chain derivatives, a few α,α disubstituted, N-methyl and α-hydroxy derivatives, but no β-amino acids. We show that over 50 unnatural amino acids can be incorporated into peptides by ribosomal translation. Using a set of analogs that are efficiently charged and translated we were able to prepare individual peptides containing up to 13 different unnatural amino acids.Conclusions/SignificanceOur results demonstrate that a diverse array of unnatural building blocks can be translationally incorporated into peptides. These building blocks provide new opportunities for in vitro selections with highly modified drug-like peptides.

Human IL-10 (hIL-10) modulates critical immune and inflammatory responses by way of interactions with its high-(IL-10R1) and low-affinity (IL-10R2) cell surface receptors. Human cytomegalovirus exploits the IL-10 signaling pathway by expressing a functional viral IL-10 homolog (cmvIL-10), which shares only 27% sequence identity with hIL-10 yet signals through IL-10R1 and IL-10R2. To define the molecular basis of this virus-host interaction, we determined the 2.7-Å crystal structure of cmvIL-10 bound to the extracellular fragment of IL-10R1 (sIL-10R1). The structure reveals cmvIL-10 forms a disulfide-linked homodimer that binds two sIL-10R1 molecules. Although cmvIL-10 and hIL-10 share similar intertwined topologies and sIL-10R1 binding sites, their respective interdomain angles differ by ϳ40°. This difference results in a striking re-organization of the IL-10R1s in the putative cell surface complex. Solution binding studies show cmvIL-10 and hIL-10 share essentially identical affinities for sIL-10R1 whereas the EpsteinBarr virus IL-10 homolog (ebvIL-10), whose structure is highly similar to hIL-10, exhibits a ϳ20-fold reduction in sIL-10R1 affinity. Our results suggest cmvIL-10 and ebvIL-10 have evolved different molecular mechanisms to engage the IL-10 receptors that ultimately enhance the respective ability of their virus to escape immune detection.

There is a great demand for the discovery of new therapeutic molecules that combine the high specificity and affinity of biologic drugs with the bioavailability and lower cost of small molecules. Small, natural-product-like peptides hold great promise in bridging this gap; however, access to libraries of these compounds has been a limitation. Since ribosomal peptides may be subjected to in vitro selection techniques, the generation of extremely large libraries (>1013) of highly modified macrocyclic peptides may provide a powerful alternative for the generation and selection of new useful bioactive molecules. Moreover, the incorporation of many non-proteinogenic amino acids into ribosomal peptides in conjunction with macrocyclization should enhance the drug-like features of these libraries. Here we show that mRNA-display, a technique that allows the in vitro selection of peptides, can be applied to the evolution of macrocyclic peptides that contain a majority of unnatural amino acids. We describe the isolation and characterization of two such unnatural cyclic peptides that bind the protease thrombin with low nanomolar affinity, and we show that the unnatural residues in these peptides are essential for the observed high-affinity binding. We demonstrate that the selected peptides are tight-binding inhibitors of thrombin, with Kiapp values in the low nanomolar range. The ability to evolve highly modified macrocyclic peptides in the laboratory is the first crucial step toward the facile generation of useful molecular reagents and therapeutic lead molecules that combine the advantageous features of biologics with those of small-molecule drugs.

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Interleukin-22 (IL-22) is a cellular homolog of IL-10 that stimulates the production of acute-phase reactants. IL-22 and IL-10 require different ligand-specific receptor chains (IL-22R and IL-10R1) but share a second receptor chain (IL-10R2) to initiate cellular responses. The quaternary structures and the ability of IL-22 and IL-10 to engage soluble (s) IL-10R1, IL-22R, IL-10R2 receptor chains were analyzed using size exclusion chromatography and surface plasmon resonance techniques. In contrast to IL-10, which is a homodimer, IL-22 is a monomer in solution that forms a 1:1 interaction with sIL-22R. Kinetic binding data reveal sIL-22R and sIL-10R1 exhibit specific nanomolar binding constants for IL-22 (k(on)/k(off) = 14.9 nM) and a monomeric isomer of IL-10 (IL-10M1) (k(on)/k(off) = 0.7 nM), respectively. In contrast, IL-10R2 exhibits essentially no affinity for IL-22 (K(eq) approximately 1 mM) or IL-10M1 (K(eq) approximately 2 mM) alone but displays a substantial increase in affinity for the IL-10/sIL-10R1 (K(eq) approximately 350 microM) and IL-22/sIL-22R (K(eq) approximately 45 microM) complexes. Three-dimensional models of IL-22 and IL-10 receptor complexes suggest two receptor residues (Gly-44 and Arg-96) are largely responsible for the marked differences in ligand affinity observed for sIL-10R1 and sIL-22R vs. sIL-10R2.