scholarly journals Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR

2021 ◽  
Vol 77 (10) ◽  
Author(s):  
Enrico Luchinat ◽  
Letizia Barbieri ◽  
Matteo Cremonini ◽  
Matteo Pennestri ◽  
Alessio Nocentini ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Dissociation Constants ◽  
Attrition Rates ◽  
Cellular Environment ◽  
Competition Binding ◽  
Intracellular Target ◽  
Intracellular Binding ◽  
Target Binding

Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding.

scholarly journals Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

2004 ◽  
Vol 186 (18) ◽  
pp. 6254-6264 ◽  
Author(s):  
Peggy Freede ◽  
Sabine Brantl
Keyword(s):  
Binding Affinity ◽  
Copy Number ◽  
Dissociation Constants ◽  
Mobility Shift ◽  
Copy Numbers ◽  
Maximal Binding ◽  
Equilibrium Dissociation Constants ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

scholarly journals Molecular insights into the copper-sensitive operon repressor in Acidithiobacillus caldus

2021 ◽  
Author(s):  
Shaoxiang Hou ◽  
Yanjun Tong ◽  
Hailin Yang ◽  
Shoushuai Feng
Keyword(s):  
Binding Affinity ◽  
Dissociation Constants ◽  
Copper Ions ◽  
Copper Resistance ◽  
Size Exclusion ◽  
Transcriptional Analysis ◽  
Copper Stress ◽  
Acidithiobacillus Caldus ◽  
Competition Assays

The copper-sensitive operon repressor (CsoR) family is the main Cu(I)-sensing family, which is widely distributed, and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥ 3 g/L copper ions). Here, we identified CsoR Ac in hyper-copper-resistant Acidithiobacillus caldus , a type strain used in the bioleaching process of copper ores. CsoR Ac possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli . Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoR Ac monomer in vitro combined with atomic absorption spectroscopy analysis. Cu I -CsoR Ac and apo-CsoR Ac share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants ( K D = 2.26 × 10 −18 M and 0.53 × 10 −15 M) and Cu(I) binding affinity of apo-CsoR Ac were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoR Ac did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants shared the same order of magnitude of Cu(I) binding affinity with apo-CsoR Ac . Moreover, apo-CsoR Ac could bind to the DNA fragment P08430 in vitro , although with low affinity. Finally, a working model was proposed to illustrate putative copper resistance mechanisms in A. caldus . Importance Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus . Here, we characterized a major Cu(I)-sensing family protein, CsoR Ac , which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator, bathocuproine disulfonate. In particular, CsoR family proteins were only identified in A. caldus , rather than A. ferrooxidans and A. thiooxidans , which are both type strains used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

scholarly journals Overview of the SAMPL6 pKa Challenge: Evaluating small molecule microscopic and macroscopic pKa predictions

2020 ◽  
Author(s):  
Mehtap Işık ◽  
Ariën S. Rustenburg ◽  
Andrea Rizzi ◽  
M. R. Gunner ◽  
David L. Mobley ◽  
...  
Keyword(s):  
Free Energy ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Dissociation Constants ◽  
Binding Free Energy ◽  
Acid Dissociation ◽  
Prediction Methods ◽  
Protonation State

AbstractThe prediction of acid dissociation constants (pKa) is a prerequisite for predicting many other properties of a small molecule, such as its protein-ligand binding affinity, distribution coefficient (log D), membrane permeability, and solubility. The prediction of each of these properties requires knowledge of the relevant protonation states and solution free energy penalties of each state. The SAMPL6 pKa Challenge was the first time that a separate challenge was conducted for evaluating pKa predictions as part of the Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) exercises. This challenge was motivated by significant inaccuracies observed in prior physical property prediction challenges, such as the SAMPL5 log D Challenge, caused by protonation state and pKa prediction issues. The goal of the pKa challenge was to assess the performance of contemporary pKa prediction methods for drug-like molecules. The challenge set was composed of 24 small molecules that resembled fragments of kinase inhibitors, a number of which were multiprotic. Eleven research groups contributed blind predictions for a total of 37 pKa distinct prediction methods. In addition to blinded submissions, four widely used pKa prediction methods were included in the analysis as reference methods. Collecting both microscopic and macroscopic pKa predictions allowed in-depth evaluation of pKa prediction performance. This article highlights deficiencies of typical pKa prediction evaluation approaches when the distinction between microscopic and macroscopic pKas is ignored; in particular, we suggest more stringent evaluation criteria for microscopic and macroscopic pKa predictions guided by the available experimental data. Top-performing submissions for macroscopic pKa predictions achieved RMSE of 0.7-1.0 pKa units and included both quantum chemical and empirical approaches, where the total number of extra or missing macroscopic pKas predicted by these submissions were fewer than 8 for 24 molecules. A large number of submissions had RMSE spanning 1-3 pKa units. Molecules with sulfur-containing heterocycles or iodo and bromo groups were less accurately predicted on average considering all methods evaluated. For a subset of molecules, we utilized experimentally-determined microstates based on NMR to evaluate the dominant tautomer predictions for each macroscopic state. Prediction of dominant tautomers was a major source of error for microscopic pKa predictions, especially errors in charged tautomers. The degree of inaccuracy in pKa predictions observed in this challenge is detrimental to the protein-ligand binding affinity predictions due to errors in dominant protonation state predictions and the calculation of free energy corrections for multiple protonation states. Underestimation of ligand pKa by 1 unit can lead to errors in binding free energy errors up to 1.2 kcal/mol. The SAMPL6 pKa Challenge demonstrated the need for improving pKa prediction methods for drug-like molecules, especially for challenging moieties and multiprotic molecules.

scholarly journals A mutant form of ERα associated with estrogen insensitivity affects the coupling between ligand binding and coactivator recruitment

2020 ◽  
Vol 13 (650) ◽  
pp. eaaw4653
Author(s):  
Yin Li ◽  
Laurel A. Coons ◽  
René Houtman ◽  
Kathryn E. Carlson ◽  
Teresa A. Martin ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Transcriptional Response ◽  
Estrogen Receptor Α ◽  
Mutant Form ◽  
Missense Mutations ◽  
Gene Encoding ◽  
Molecular Features ◽  
Transcriptional Regulatory

A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα–coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.

Microfluidic Molecular Trap: Probing Extracellular Signaling by Selectively Blocking Exchange of Specific Molecules in Cell-Cell Interactions

2013 ◽  
Author(s):  
Bryson M. Brewer ◽  
Yandong Gao ◽  
Rebecca M. Sappington ◽  
Deyu Li
Keyword(s):  
Cell Culture ◽  
Signaling Pathway ◽  
Binding Affinity ◽  
Dissociation Constants ◽  
Gene Knockout ◽  
In Vitro Cell Culture ◽  
Extracellular Signaling ◽  
Time Periods ◽  
Avidin Biotin Complex

Communication among cell populations is achieved via a wide variety of soluble, extracellular signaling molecules [1]. In order to investigate the role of specific molecules in a cellular process, researchers often utilize in vitro cell culture techniques in which the molecule under question has been removed from the signaling pathway. Traditionally, this has been accomplished by eliminating the gene in the cell that is responsible for coding the targeted ligand/receptor by using modern DNA technology such as gene knockout; however, this process is expensive, time-consuming, and labor intensive. Previously, we have demonstrated a microfluidic platform that uses a semi-permeable barrier with embedded receptor-coated nanoparticles to selectively remove a specific molecule or ligand from the extracellular signaling pathway in a cell co-culture environment [2]. This initial proof-of-principle was conducted using biotinylated nanoparticles and fluorescently tagged avidin molecules, as the avidin/biotin complex is the strongest known non-covalent interaction between a protein and a ligand (Dissociation constant kd = 10−15 M). Also, the trap was only effective for short time periods (<15 min) because the high concentration of fluorescently tagged avidin molecules required for visualization quickly saturated the barrier. However, nearly all biologically relevant ligand-receptor interactions have lower binding affinities than the avidin-biotin complex, with dissociation constants that are larger by several orders of magnitude. In addition, many in vitro cell culture experiments are conducted over multiple hours or days. Thus, a practically useful molecular trap device must be able to operate in a lower binding affinity regime while also lasting for extended time periods. Here we present results in which a biotinylated-particle barrier was used to successfully block lower concentrations of fluorescently tagged avidin for multiple days, showcasing the applicability of the device for long term experiments. In addition, we introduce a modified molecular trap in which the protein A/goat IgG complex was used to demonstrate the effectiveness of the platform for lower binding affinity protein-ligand interactions. These results indicate the potential usefulness of the microfluidic molecular trap platform for probing extracellular signaling pathways.

scholarly journals Characterization of ligand binding to acyl-CoA-binding protein

1993 ◽  
Vol 290 (2) ◽  
pp. 321-326 ◽  
Author(s):  
J Rosendal ◽  
P Ertbjerg ◽  
J Knudsen
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Intracellular Transport ◽  
Binding Protein ◽  
Acyl Chain ◽  
Head Group ◽  
Binding Studies ◽  
Competition Binding ◽  
Chain Acyl ◽  
Sepharose 4B

Ligand binding to recombinant bovine acyl-CoA-binding protein (rACBP) was examined using a Lipidex 1000 competition assay and an e.p.r. spectroscopy displacement assay. Of all putative ligands tested, rACBP exhibited a high binding affinity only for acyl-CoA esters. No alternative ligands could be found in rat liver fractions purified on an affinity of column on which ACBP was coupled to Sepharose 4B. E.p.r. data indicate that both the acyl chain and the CoA head group of acyl-CoA are involved in binding and that the 3′-phosphate group on the ribose moiety of acyl-CoA esters plays a crucial role in the binding of acyl-CoA to ACBP. E.p.r. competition binding studies show that the binding affinity of acyl-CoA esters for rACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-CoA esters with 14-22 carbon atoms in the acyl chain. No correlation between the number of double bonds in the acyl chain and the binding affinity was observed. The experimental results strongly indicate that ACBP specifically binds long-chain acyl-CoA esters with a very high affinity, results that indicate that ACBP is likely to be involved in the intracellular transport and pool formation of these compounds.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Automated, Accurate, and Scalable Relative Protein-Ligand Binding Free Energy Calculations using Lambda Dynamics

2020 ◽  
Author(s):  
E. Prabhu Raman ◽  
Thomas J. Paul ◽  
Ryan L. Hayes ◽  
Charles L. Brooks III
Keyword(s):  
Free Energy ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Binding Free Energy ◽  
Computational Cost ◽  
Combinatorial Libraries ◽  
Free Energy Calculations ◽  
Lead Optimization ◽  
Efficient Estimation ◽  
Lead Compound

<p>Accurate predictions of changes to protein-ligand binding affinity in response to chemical modifications are of utility in small molecule lead optimization. Relative free energy perturbation (FEP) approaches are one of the most widely utilized for this goal, but involve significant computational cost, thus limiting their application to small sets of compounds. Lambda dynamics, also rigorously based on the principles of statistical mechanics, provides a more efficient alternative. In this paper, we describe the development of a workflow to setup, execute, and analyze Multi-Site Lambda Dynamics (MSLD) calculations run on GPUs with CHARMm implemented in BIOVIA Discovery Studio and Pipeline Pilot. The workflow establishes a framework for setting up simulation systems for exploratory screening of modifications to a lead compound, enabling the calculation of relative binding affinities of combinatorial libraries. To validate the workflow, a diverse dataset of congeneric ligands for seven proteins with experimental binding affinity data is examined. A protocol to automatically tailor fit biasing potentials iteratively to flatten the free energy landscape of any MSLD system is developed that enhances sampling and allows for efficient estimation of free energy differences. The protocol is first validated on a large number of ligand subsets that model diverse substituents, which shows accurate and reliable performance. The scalability of the workflow is also tested to screen more than a hundred ligands modeled in a single system, which also resulted in accurate predictions. With a cumulative sampling time of 150ns or less, the method results in average unsigned errors of under 1 kcal/mol in most cases for both small and large combinatorial libraries. For the multi-site systems examined, the method is estimated to be more than an order of magnitude more efficient than contemporary FEP applications. The results thus demonstrate the utility of the presented MSLD workflow to efficiently screen combinatorial libraries and explore chemical space around a lead compound, and thus are of utility in lead optimization.</p>

Sign in / Sign up

Export Citation Format

Share Document