3ltq | pdb_00003ltq
Structure of Interleukin 1B solved by SAD using an inserted Lanthanide Binding Tag
Structural highlights
Function[IL1B_HUMAN] Produced by activated macrophages, IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. IL-1 proteins are involved in the inflammatory response, being identified as endogenous pyrogens, and are reported to stimulate the release of prostaglandin and collagenase from synovial cells.[1] Publication Abstract from PubMedLanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1beta (IL1beta) and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb(3+) tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1beta-S1 and IL1beta-L3 constructs and for the remaining constructs by comparing the (1)H-(15)N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1beta. Additionally, binding of LBT-loop IL1beta proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1beta structure. The paramagnetic NMR spectra of loop-LBT mutant IL1beta-R2 were assigned and the Deltachi tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1beta-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling. Engineering Encodable Lanthanide-Binding Tags into Loop Regions of Proteins.,Barthelmes K, Reynolds AM, Peisach E, Jonker HR, Denunzio NJ, Allen KN, Imperiali B, Schwalbe H J Am Chem Soc. 2010 Dec 23. PMID:21182275[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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