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1.
Nat Commun ; 15(1): 3105, 2024 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-38600130

RESUMEN

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.


Asunto(s)
Proteínas Intrínsecamente Desordenadas , Vibrio cholerae , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vibrio cholerae/metabolismo , Dispersión del Ángulo Pequeño , Unión Proteica , Difracción de Rayos X , ADN/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo
3.
Int J Biol Macromol ; 182: 502-511, 2021 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-33848543

RESUMEN

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation. In parallel, the complex was cross-linked, digested and the resulting peptides were characterized by mass-spectrometry to define maximal distance restraints between HER2 and A10 amino acids in their complex. These independent datasets guided a docking process, refined by molecular dynamics simulations, to develop a model of the complex and estimate per-residue free-energy contributions. Such a model explains the experimental data and identifies a second, non-canonical paratope, located in the region opposite to the conventional nanobody paratope, formed by the hypervariable loop regions LH1 and LH3. Both paratopes contributed substantially to the overall affinity by binding to independent HER2 epitopes. Nanobody mutants with substitution of key interaction residues, as indicated by the model, possess significantly lower affinity for HER2. This is the first described case of a "natural" biparatopic nanobody, directly selected by in-vitro panning.


Asunto(s)
Sitios de Unión de Anticuerpos , Receptor ErbB-2/química , Anticuerpos de Cadena Única/química , Humanos , Simulación del Acoplamiento Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Unión Proteica , Receptor ErbB-2/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología
4.
Proc Natl Acad Sci U S A ; 117(52): 32891-32901, 2020 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-33323484

RESUMEN

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant's hierarchical assembly, and establish a direct correlation between the protein's structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells' color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

5.
Nucleic Acids Res ; 48(7): 3975-3986, 2020 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-32095808

RESUMEN

Guanine-rich regions of the human genome can adopt non-canonical secondary structures. Their role in regulating gene expression has turned them into promising targets for therapeutic intervention. Ligands based on polyaromatic moieties are especially suitable for targeting G-quadruplexes utilizing their size complementarity to interact with the large exposed surface area of four guanine bases. A predictable way of (de)stabilizing specific G-quadruplex structures through efficient base stacking of polyaromatic functional groups could become a valuable tool in our therapeutic arsenal. We have investigated the effect of pyrene-modified uridine nucleotides incorporated at several positions of the thrombin binding aptamer (TBA) as a model system. Characterization using spectroscopic and biophysical methods provided important insights into modes of interaction between pyrene groups and the G-quadruplex core as well as (de)stabilization by enthalpic and entropic contributions. NMR data demonstrated that incorporation of pyrene group into G-rich oligonucleotide such as TBA may result in significant changes in 3D structure such as formation of novel dimeric topology. Site specific structural changes induced by stacking of the pyrene moiety on nearby nucleobases corelate with distinct thrombin binding affinities and increased resistance against nuclease degradation.


Asunto(s)
Aptámeros de Nucleótidos/química , G-Cuádruplex , Pirenos/química , Aptámeros de Nucleótidos/sangre , Aptámeros de Nucleótidos/metabolismo , Desoxirribonucleasas , Dimerización , Entropía , Humanos , Termodinámica , Trombina/metabolismo , Nucleótidos de Uracilo/química
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