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Constructing protein polyhedra via orthogonal chemical interactions.

ABSTRACT: Many proteins exist naturally as symmetrical homooligomers or homopolymers1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design2-5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures1,6-a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein-protein interactions and symmetry are simultaneously achieved by selective, 'one-pot' coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe3+ and Zn2+ coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures7,8 and are, to our knowledge, unique among designed systems9-13 in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design.

SUBMITTER: Golub E

PROVIDER: S-EPMC7007351 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Constructing protein polyhedra via orthogonal chemical interactions.

Golub Eyal E Subramanian Rohit H RH Esselborn Julian J Alberstein Robert G RG Bailey Jake B JB Chiong Jerika A JA Yan Xiaodong X Booth Timothy T Baker Timothy S TS Tezcan F Akif FA

Nature 20200122 7793

Many proteins exist naturally as symmetrical homooligomers or homopolymers1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design2-5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures1,6-a daunting task for prot ...[more]

PMID: 31969701

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Project description:The specificity of the kinase-regulator interaction is driven by a limited set of interfacial residues in each protein that strongly. To identify combinations of these interface residues that are functional and potentially insulated from existing two-component signaling pathways in E. coli, we constructed a dual library of mutants in which the key, coevolving interface residues of a canonical two-component system, PhoQ and PhoP, were randomized. We used NNS codons to randomize six residues in PhoQ and five residues in PhoP, all of which lie at the interface formed by the two proteins in complex and that are critical to determining partner specificity in all two-component signaling pathways. To identify functional combinations of residues, we first grew the library of PhoQ-PhoP variants overnight in medium with low Mg2+, which activates PhoQ. Because cells must phosphorylate PhoP to grow when extracellular Mg2+ is limiting, this step enriches for functional PhoQ-PhoP variants. Variants that survived selection in limiting Mg2+ were then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin. We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.

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Constructing protein polyhedra via orthogonal chemical interactions.

Publications

Constructing protein polyhedra via orthogonal chemical interactions.

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