Probing coupled conformational transitions of intrinsically disordered proteins in their interactions with target proteins

Intrinsically disordered proteins or regions (IDPs or IDRs) are abundant in the eukaryotic proteome and critical in regulation of dynamic cellular processes. Intensive structural investigations have proposed the molecular mechanisms of the interaction between IDRs and their binding partners. Here we extract the distinct thermodynamic features of coupled conformational transitions of IDRs founding the interaction mechanisms. We also present simulation tools to facilitate a design of the calorimetric experiments probing and quantifying the conformational transitions of IDRs. The suggested thermodynamic approach will further advance our understanding of distribution among multiple states of IDRs in their interactions with target molecules.N

Quantitative Frameworks for Multivalent Macromolecular Interactions in Biological Linear Lattice Systems

Multivalent macromolecular interactions underlie dynamic regulation of diverse biological processes in ever-changing cellular states. These interactions often involve binding of multiple proteins to a linear lattice including intrinsically disordered proteins and the chromosomal DNA with many repeating recognition motifs. Quantitative understanding of such multivalent interactions on a linear lattice is crucial for exploring their unique regulatory potentials in the protein interactions. To this end, we present two specific examples where the quantitative models have been applied and further extended to provide biological insights into specific cellular processes. In the first case, the conditional probability model was extended to highlight the significant impact of nonspecific binding of transcription factors to the chromosomal DNA on gene-specific transcriptional activities. The second case presents the recently developed combinatorial models to unravel the complex organization of target protein binding sites within an intrinsically disordered region (IDR) of a nucleoporin. In particular, these models have suggested a unique function of IDRs as a molecular switch coupling distinct cellular processes. The quantitative models reviewed here are envisioned to further advance for dissection and functional studies of more complex systems including phase-separated biomolecular condensates.N

Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein of Escherichia coli▿

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 Å) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues

A C-helix residue, Arg-123, has important roles in both the active and inactive forms of the cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper β4/β5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously (Berman, H. M., Ten Eyck, L. F., Goodsell, D. S., Haste, N. M., Korney, A., and Taylor, S. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 45-50). Based on mutagenic evidence, we also propose the basis for the stabilization of the inactive form to be through a salt interaction between Asp-68 and Arg-123. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.OAIID:RECH_ACHV_DSTSH_NO:T200700352RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A080565CITE_RATE:5.581DEPT_NM:생명과학부EMAIL:[email protected]_YN:YY

Thermodynamic Models for Assembly of Intrinsically Disordered Protein Hubs with Multiple Interaction Partners

Prevalent in diverse protein interactomes, intrinsically disordered proteins or regions (IDPs or IDRs) often drive assembly of higher-order macromolecular complexes, using multiple target-binding motifs. Such IDP hubs are suggested to process various cellular signals and coordinate relevant biological processes. However, the mechanism of assembly and functional regulation of IDP hubs remains elusive due to the challenges in dissecting their intricate protein-protein interaction networks. Here we present basic thermodynamic models for the assembly of simple IDP hubs with multiple target proteins, constructing partition functions from fundamental binding parameters. We combined these basic functions to develop advanced thermodynamic models to analyze the assembly of the Nup153 hubs interacting with multiple karyopherin beta 1 (Kap) molecules, critical components of nucleocytoplasmic transport. The thermodynamic analysis revealed a complex organization of the Kap binding sites within the C-terminal IDR of Nup153 including a high-affinity 1:1 interaction site and a series of low-affinity sites for binding of multiple Kaps with negative cooperativity. The negative cooperativity arises from the overlapping nature of the low-affinity sites where Kap occupies multiple dipeptide motifs. The quantitative dissection culminated in construction of the Nup153 hub ensemble, elucidating how distribution among various Kap-bound states is modulated by Kap concentration and competing nuclear proteins. In particular, the Kap occupancy of the IDR can be fine-tuned by varying the location of competition within the overlapping sites, suggesting coupling of specific nuclear processes to distinct transport activities. In general, our results demonstrate the feasibility and a potential mechanism for manifold regulation of IDP functions by diverse cellular signals.N

Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification

Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons