RESUMEN
Fragment-based drug design is heavily dependent on the optimization of initial low-affinity binders. Herein we introduce an approach that uses selective labeling of methyl groups in leucine and isoleucine side chains to directly probe methyl-π contacts, one of the most prominent forms of interaction between proteins and small molecules. Using simple NMR chemical shift perturbation experiments with selected BRD4-BD1 binders, we find good agreement with a commonly used model of the ring-current effect as well as the overall interaction geometries extracted from the Protein Data Bank. By combining both interaction geometries and chemical shift calculations as fit quality criteria, we can position dummy aromatic rings into an AlphaFold model of the protein of interest. The proposed method can therefore provide medicinal chemists with important information about binding geometries of small molecules in fast and iterative matter, even in the absence of high-resolution experimental structures.
Asunto(s)
Modelos Moleculares , Ligandos , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Unión Proteica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas/química , Proteínas/metabolismo , Diseño de Fármacos , Espectroscopía de Resonancia Magnética , Proteínas que Contienen BromodominioRESUMEN
Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.
Asunto(s)
Proteínas Nucleares , Valina , Leucina/química , Valina/química , Ligandos , Factores de TranscripciónRESUMEN
In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13C-1 H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13C-T2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.
Asunto(s)
Isoleucina , Factores de Transcripción , Factores de Transcripción/química , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligandos , Resonancia Magnética Nuclear BiomolecularRESUMEN
The availability of high-resolution 3D structural information is crucial for investigating guest-host systems across a wide range of fields. In the context of drug discovery, the information is routinely used to establish and validate structure-activity relationships, grow initial hits from screening campaigns, and to guide molecular docking. For the generation of protein-ligand complex structural information, X-ray crystallography is the experimental method of choice, however, with limited information on protein flexibility. An experimentally verified structural model of the binding interface in the native solution-state would support medicinal chemists in their molecular design decisions. Here we demonstrate that protein-bound ligand 1 H NMR chemical shifts are highly sensitive and accurate probes for the immediate chemical environment of protein-ligand interfaces. By comparing the experimental ligand 1 H chemical shift values with those computed from the X-ray structure using quantum mechanics methodology, we identify significant disagreements for parts of the ligand between the two experimental techniques. We show that quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) ensembles can be used to refine initial X-ray co-crystal structures resulting in a better agreement with experimental 1 H ligand chemical shift values. Overall, our findings highlight the usefulness of ligand 1 H NMR chemical shift information in combination with a QM/MM MD workflow for generating protein-ligand ensembles that accurately reproduce solution structural data.
Asunto(s)
Imagen por Resonancia Magnética , Proteínas , Simulación del Acoplamiento Molecular , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Proteínas/químicaRESUMEN
In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.
RESUMEN
Aberrant WNT pathway activation, leading to nuclear accumulation of ß-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of ß-catenin and subsequent nuclear translocation. Restoring cellular degradation of ß-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to ß-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging ß-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards ß-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/química , beta Catenina/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , beta Catenina/metabolismoRESUMEN
Loss of WRN, a DNA repair helicase, was identified as a strong vulnerability of microsatellite instable (MSI) cancers, making WRN a promising drug target. We show that ATP binding and hydrolysis are required for genome integrity and viability of MSI cancer cells. We report a 2.2-Å crystal structure of the WRN helicase core (517-1,093), comprising the two helicase subdomains and winged helix domain but not the HRDC domain or nuclease domains. The structure highlights unusual features. First, an atypical mode of nucleotide binding that results in unusual relative positioning of the two helicase subdomains. Second, an additional ß-hairpin in the second helicase subdomain and an unusual helical hairpin in the Zn2+ binding domain. Modelling of the WRN helicase in complex with DNA suggests roles for these features in the binding of alternative DNA structures. NMR analysis shows a weak interaction between the HRDC domain and the helicase core, indicating a possible biological role for this association. Together, this study will facilitate the structure-based development of inhibitors against WRN helicase.
Asunto(s)
Dominio Catalítico , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Inestabilidad de Microsatélites , Helicasa del Síndrome de Werner/química , Helicasa del Síndrome de Werner/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/genética , Supervivencia Celular/genética , Cristalización , ADN/metabolismo , Daño del ADN/genética , Silenciador del Gen , Células HCT116 , Humanos , Hidrólisis , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transfección , Zinc/metabolismo , Quinasa Tipo Polo 1RESUMEN
While CH-π interactions with target proteins are crucial determinants for the affinity of arguably every drug molecule, no method exists to directly measure the strength of individual CH-π interactions in drug-protein complexes. Herein, we present a fast and reliable methodology called PI (π interactions) by NMR, which can differentiate the strength of protein-ligand CH-π interactions in solution. By combining selective amino-acid side-chain labeling with 1 H-13 Câ NMR, we are able to identify specific protein protons of side-chains engaged in CH-π interactions with aromatic ring systems of a ligand, based solely on 1 H chemical-shift values of the interacting protein aromatic ring protons. The information encoded in the chemical shifts induced by such interactions serves as a proxy for the strength of each individual CH-π interaction. PI by NMR changes the paradigm by which chemists can optimize the potency of drug candidates: direct determination of individual π interactions rather than averaged measures of all interactions.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Humanos , Modelos MolecularesRESUMEN
In recent years, we developed a toolbox of heavy isotope containing compounds, which serve as metabolic amino acid precursors in the E. coli-based overexpression of aromatic residue labeled proteins. Our labeling techniques show excellent results both in terms of selectivity and isotope incorporation levels. They are additionally distinguished by low sample production costs and meet the economic demands to further implement protein NMR spectroscopy as a routinely used method in drug development processes. Different isotopologues allow for the assembly of optimized protein samples, which fulfill the requirements of various NMR experiments to elucidate protein structures, analyze conformational dynamics, or probe interaction surfaces. In the present article, we want to summarize the precursors we developed so far and give examples of their special value in the probing of protein-ligand interaction.
Asunto(s)
Aminoácidos Aromáticos/química , Marcaje Isotópico/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Aminoácidos Aromáticos/metabolismo , Animales , Escherichia coli/metabolismo , Humanos , Ligandos , Sondas Moleculares , Proteínas/químicaRESUMEN
The application of metabolic precursors for selective stable isotope labeling of aromatic residues in cell-based protein overexpression has already resulted in numerous NMR probes to study the structural and dynamic characteristics of proteins. With anthranilic acid, we present the structurally simplest precursor for exclusive tryptophan side chain labeling. A synthetic route to 13C, 2H isotopologues allows the installation of isolated 13C-1H spin systems in the indole ring of tryptophan, representing a versatile tool to investigate side chain motion using relaxation-based experiments without the loss of magnetization due to strong 1JCC and weaker 2JCH scalar couplings, as well as dipolar interactions with remote hydrogens. In this article, we want to introduce this novel precursor in the context of hitherto existing techniques of in vivo aromatic residue labeling.
Asunto(s)
Marcaje Isotópico/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , ortoaminobenzoatos/química , Triptófano/químicaRESUMEN
Fragment-based drug design exploits initial screening of low molecular weight compounds and their concomitant affinity improvement. The multitude of possible chemical modifications highlights the necessity to obtain structural information about the binding mode of a fragment. Herein we describe a novel NMR methodology (LOGSY titration) that allows the determination of binding modes of low affinity binders in the protein-ligand interface and reveals suitable ligand positions for the addition of functional groups that either address or substitute protein-bound water, information of utmost importance for drug design. The particular benefit of the methodology and in contrast to conventional ligand-based methods is the independence of the molecular weight of the protein under study. The validity of the novel approach is demonstrated on two ligands interacting with bromodomain 1 of bromodomain containing protein 4, a prominent cancer target in pharmaceutical industry.
Asunto(s)
Diseño de Fármacos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Agua/química , Sitios de Unión , Proteínas de Ciclo Celular , Humanos , Ligandos , Modelos Moleculares , Proteínas Nucleares/química , Unión Proteica , Volumetría , Factores de Transcripción/químicaRESUMEN
The importance of NMR spectroscopy in unraveling the structural and dynamic properties of proteins is ever-expanding owing to progress in experimental techniques, hardware development, and novel labeling approaches. Multiple sophisticated methods of aliphatic residue labeling can be found in the literature, whereas the selective incorporation of NMR active isotopes into other amino acids still holds the potential for improvement. In order to close this methodological gap, we present a novel metabolic precursor for cell-based protein overexpression to assemble 13 C/2 H isotope patterns in the peptide backbone, as well as in side chain positions of a mechanistically distinguished histidine residue.
Asunto(s)
Escherichia coli/metabolismo , Histidina/química , Imidazoles/química , Marcaje Isotópico/métodos , Piruvatos/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Radioisótopos de Carbono , Deuterio , Escherichia coli/genética , Histidina/biosíntesis , Humanos , Imidazoles/metabolismo , Espectroscopía de Resonancia Magnética , Piruvatos/metabolismo , Transaminasas/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Microtubule-associated protein 1B (MAP1B) is a classical high molecular mass microtubule-associated protein expressed at high levels in the brain. It confers specific properties to neuronal microtubules and is essential for neuronal differentiation, brain development and synapse maturation. Misexpression of the protein contributes to the development of brain disorders in humans. However, despite numerous reports demonstrating the importance of MAP1B in regulation of the neuronal cytoskeleton during neurite extension and axon guidance, its mechanism of action is still elusive. Here we focus on the intrinsically disordered microtubule binding domain of the light chain of MAP1B. In order to obtain more detailed structural information about this domain we assigned NMR chemical shifts of backbone and aliphatic side chain atoms.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Secuencia de Aminoácidos , Animales , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , RatasRESUMEN
Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity and undergo rearrangements of the time-averaged conformational ensemble on changes of environmental conditions (e.g., in ionic strength, pH, molecular crowding). In contrast to stably folded proteins, IDPs often form compact conformations at acidic pH. The biological relevance of this process was, for example, demonstrated by nuclear magnetic resonance studies of the aggregation prone (low pH) state of α-synuclein. In this study, we report a large-scale analysis of the pH dependence of disordered proteins using the recently developed meta-structure approach. The meta-structure analysis of a large set of IDPs revealed a significant tendency of IDPs to form α-helical secondary structure elements and to preferentially fold into more compact structures under acidic conditions. The predictive validity of this novel approach was demonstrated with applications to the tumor-suppressor BASP1 and the transcription factor Tcf4.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Proteínas Intrínsecamente Desordenadas/química , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Protones , Proteínas Represoras/química , Factores de Transcripción/química , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Concentración Osmolar , Conformación Proteica , Factor de Transcripción 4RESUMEN
An ultra-high-resolution NMR experiment for the measurement of intraresidue (1)H(i)-(15)N(i)-(13)C'(i) dipolar-chemical shift anisotropy relaxation interference is employed to extract information about local backbone geometries in intrinsically disordered proteins. The study of tumor suppressor BASP1 revealed a population shift of ß-turn geometries at low pH conditions and a compaction of the BASP1 structural ensemble.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Simulación por Computador , Modelos Moleculares , Conformación ProteicaRESUMEN
The characterization of the conformational properties of intrinsically disordered proteins (IDPs), and their interaction modes with physiological partners has recently become a major research topic for understanding biological function on the molecular level. Although multidimensional NMR spectroscopy is the technique of choice for the study of IDPs at atomic resolution, the intrinsically low resolution, and the large peak intensity variations often observed in NMR spectra of IDPs call for resolution- and sensitivity-optimized pulse schemes. We present here a set of amide proton-detected 3D BEST-TROSY correlation experiments that yield the required sensitivity and spectral resolution for time-efficient sequential resonance assignment of large IDPs. In addition, we introduce two proline-edited 2D experiments that allow unambiguous identification of residues adjacent to proline that is one of the most abundant amino acids in IDPs. The performance of these experiments, and the advantages of BEST-TROSY pulse schemes are discussed and illustrated for two IDPs of similar length (~270 residues) but with different conformational sampling properties.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/análisis , Proteínas/química , Conformación ProteicaRESUMEN
Brain acid-soluble protein 1 (BASP1, CAP-23, NAP-22) appears to be implicated in diverse cellular processes. An N-terminally myristoylated form of BASP1 has been discovered to participate in the regulation of actin cytoskeleton dynamics in neurons, whereas non-myristoylated nuclear BASP1 acts as co-suppressor of the potent transcription regulator WT1 (Wilms' Tumor suppressor protein 1). Here we report NMR chemical shift assignment of recombinant human BASP1 fused to an N-terminal cleavable His6-tag.
Asunto(s)
Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Resonancia Magnética Nuclear Biomolecular , Protones , Proteínas Represoras/química , Secuencia de Aminoácidos , Isótopos de Carbono , Humanos , Isótopos de Nitrógeno , Estructura Secundaria de ProteínaRESUMEN
Fragment-based lead discovery (FBLD) has become a prime component of the armamentarium of modern drug design programs. FBLD identifies low molecular weight ligands that weakly bind to important biological targets. Three-dimensional structural information about the binding mode is provided by X-ray crystallography or NMR spectroscopy and is subsequently used to improve the lead compounds. Despite tremendous success rates, FBLD relies on the availability of high-resolution structural information, still a bottleneck in drug discovery programs. To overcome these limitations, we recently demonstrated that the meta-structure approach provides an alternative route to rational lead identification in cases where no 3D structure information about the biological target is available. Combined with information-rich NMR data, this strategy provides valuable information for lead development programs. We demonstrate with several examples the feasibility of the combined NMR and meta-structure approach to devise a rational strategy for fragment evolution without resorting to highly resolved protein complex structures.
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Diseño de Fármacos , Estructura Molecular , Cristalografía por Rayos X , Descubrimiento de Drogas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , beta Catenina/químicaRESUMEN
Siderocalin Q83 is a small soluble protein that has the ability to bind two different ligands (enterobactin and arachidonic acid) simultaneously in two distinct binding sites. Here we report that Q83 exhibits an intriguing dynamic behavior. In its free form, the protein undergoes significant micro-to-millisecond dynamics. When binding arachidonic acid, the motions of the arachidonic acid binding site are quenched while the dynamics at the enterobactin binding site increases. Reciprocally, enterobactin binding to Q83 quenches the motions at the enterobactin binding site and increases the slow dynamics at the arachidonic acid binding site. Additionally, in the enterobactin-bound state, the excited state of the arachidonic acid binding site resembles the arachidonic acid-bound state. These observations strongly suggest an allosteric regulation where binding of one ligand enhances the affinity of Q83 for the other one. Additionally, our data strengthen the emerging view of proteins as dynamic ensembles interconverting between different sub-states with distinct functionalities.