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1.
Am J Hum Genet ; 108(7): 1283-1300, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214447

RESUMO

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Análise Mutacional de DNA , Diploide , Biblioteca Gênica , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/fisiologia , Saccharomyces cerevisiae/genética
2.
Nucleic Acids Res ; 48(11): 6382-6402, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383734

RESUMO

The Cys2His2 zinc finger is the most common DNA-binding domain expanding in metazoans since the fungi human split. A proposed catalyst for this expansion is an arms race to silence transposable elements yet it remains poorly understood how this domain is able to evolve the required specificities. Likewise, models of its DNA binding specificity remain error prone due to a lack of understanding of how adjacent fingers influence each other's binding specificity. Here, we use a synthetic approach to exhaustively investigate binding geometry, one of the dominant influences on adjacent finger function. By screening over 28 billion protein-DNA interactions in various geometric contexts we find the plasticity of the most common natural geometry enables more functional amino acid combinations across all targets. Further, residues that define this geometry are enriched in genomes where zinc fingers are prevalent and specificity transitions would be limited in alternative geometries. Finally, these results demonstrate an exhaustive synthetic screen can produce an accurate model of domain function while providing mechanistic insight that may have assisted in the domains expansion.


Assuntos
Modelos Moleculares , Domínios Proteicos/fisiologia , Dedos de Zinco/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/síntese química , DNA/genética , DNA/metabolismo , Aprendizado Profundo , Humanos , Ligação de Hidrogênio , Domínios Proteicos/genética , Reprodutibilidade dos Testes , Especificidade por Substrato/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética
3.
Proteins ; 87(3): 236-244, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30520126

RESUMO

Peptide-based therapeutics are an alternative to small molecule drugs as they offer superior specificity, lower toxicity, and easy synthesis. Here we present an approach that leverages the dramatic performance increase afforded by the recent arrival of GPU accelerated thermodynamic integration (TI). GPU TI facilitates very fast, highly accurate binding affinity optimization of peptides against therapeutic targets. We benchmarked TI predictions using published peptide binding optimization studies. Prediction of mutations involving charged side-chains was found to be less accurate than for non-charged, and use of a more complex 3-step TI protocol was found to boost accuracy in these cases. Using the 3-step protocol for non-charged side-chains either had no effect or was detrimental. We use the benchmarked pipeline to optimize a peptide binding to our recently discovered cancer target: EME1. TI calculations predict beneficial mutations using both canonical and non-canonical amino acids. We validate these predictions using fluorescence polarization and confirm that binding affinity is increased. We further demonstrate that this increase translates to a significant reduction in pancreatic cancer cell viability.


Assuntos
Endodesoxirribonucleases/química , Neoplasias Pancreáticas/tratamento farmacológico , Peptídeos/química , Termodinâmica , Aminoácidos/química , Sobrevivência Celular/efeitos dos fármacos , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/genética , Humanos , Simulação de Dinâmica Molecular , Mutação/genética , Neoplasias Pancreáticas/genética , Peptídeos/genética , Peptídeos/farmacologia , Ligação Proteica
4.
Artigo em Inglês | MEDLINE | ID: mdl-29735742

RESUMO

Many functional roles have been attributed to homodimers, the most common mode of protein self-association, notably in the regulation of enzymes, ion channels, transporters and transcription factors. Here we review findings that offer new insights into the different roles conformational flexibility plays in regulating homodimer function. Intertwined homodimers of two-domain proteins and their related family members display significant conformational flexibility, which translates into concerted motion between structural domains. This flexibility enables the corresponding proteins to regulate function across family members by modulating the spatial positions of key recognition surfaces of individual domains, to either maintain subunit interfaces, alter them or break them altogether, leading to a variety of functional consequences. Many proteins may exist as monomers but carry out their biological function as homodimers or higher-order oligomers. We present early evidence that in such systems homodimer formation primes the protein for its functional role. It does so by inducing elevated mobility in protein regions corresponding to the binding epitopes of functionally important ligands. In some systems this process acts as an allosteric response elicited by the self-association reaction itself. Our analysis furthermore suggests that the induced extra mobility likely facilitates ligand binding through the mechanism of conformational selection.This article is part of a discussion meeting issue 'Allostery and molecular machines'.


Assuntos
Regulação Alostérica , Proteínas de Bactérias/química , Conformação Proteica , Modelos Moleculares , Ligação Proteica
5.
Proc Natl Acad Sci U S A ; 115(7): 1505-1510, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29378946

RESUMO

Biologics are a rapidly growing class of therapeutics with many advantages over traditional small molecule drugs. A major obstacle to their development is that proteins and peptides are easily destroyed by proteases and, thus, typically have prohibitively short half-lives in human gut, plasma, and cells. One of the most effective ways to prevent degradation is to engineer analogs from dextrorotary (D)-amino acids, with up to 105-fold improvements in potency reported. We here propose a general peptide-engineering platform that overcomes limitations of previous methods. By creating a mirror image of every structure in the Protein Data Bank (PDB), we generate a database of ∼2.8 million D-peptides. To obtain a D-analog of a given peptide, we search the (D)-PDB for similar configurations of its critical-"hotspot"-residues. As a proof of concept, we apply our method to two peptides that are Food and Drug Administration approved as therapeutics for diabetes and osteoporosis, respectively. We obtain D-analogs that activate the GLP1 and PTH1 receptors with the same efficacy as their natural counterparts and show greatly increased half-life.


Assuntos
Aminoácidos/química , Bases de Dados de Proteínas , Peptídeos/química , Engenharia de Proteínas/métodos , Algoritmos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células HEK293 , Meia-Vida , Humanos , Hormônio Paratireóideo/agonistas , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacocinética , Conformação Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Reprodutibilidade dos Testes
6.
PLoS One ; 12(11): e0187524, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29108013

RESUMO

Redesigning protein surface topology to improve target binding holds great promise in the search for highly selective therapeutics. While significant binding improvements can be achieved using natural amino acids, the introduction of non-canonical residues vastly increases sequence space and thus the chance to significantly out-compete native partners. The potency of protein inhibitors can be further enhanced by synthesising mirror image, D-amino versions. This renders them non-immunogenic and makes them highly resistant to proteolytic degradation. Current experimental design methods often preclude the use of D-amino acids and non-canonical amino acids for a variety of reasons. To address this, we build an in silico pipeline for D-protein designs featuring non-canonical amino acids. For a test scaffold we use an existing D-protein inhibitor of VEGF: D-RFX001. We benchmark the approach by recapitulating previous experimental optimisation with canonical amino acids. Subsequent incorporation of non-canonical amino acids allows designs that are predicted to improve binding affinity by up to -7.18 kcal/mol.


Assuntos
Aminoácidos/química , Proteínas/química , Sequência de Aminoácidos , Proteínas/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
7.
Genome Biol ; 18(1): 167, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28877740

RESUMO

BACKGROUND: The C2H2 zinc finger (C2H2-ZF) is the most numerous protein domain in many metazoans, but is not as frequent or diverse in other eukaryotes. The biochemical and evolutionary mechanisms that underlie the diversity of this DNA-binding domain exclusively in metazoans are, however, mostly unknown. RESULTS: Here, we show that the C2H2-ZF expansion in metazoans is facilitated by contribution of non-base-contacting residues to DNA binding energy, allowing base-contacting specificity residues to mutate without catastrophic loss of DNA binding. In contrast, C2H2-ZF DNA binding in fungi, plants, and other lineages is constrained by reliance on base-contacting residues for DNA-binding functionality. Reconstructions indicate that virtually every DNA triplet was recognized by at least one C2H2-ZF domain in the common progenitor of placental mammals, but that extant C2H2-ZF domains typically bind different sequences from these ancestral domains, with changes facilitated by non-base-contacting residues. CONCLUSIONS: Our results suggest that the evolution of C2H2-ZFs in metazoans was expedited by the interaction of non-base-contacting residues with the DNA backbone. We term this phenomenon "kaleidoscopic evolution," to reflect the diversity of both binding motifs and binding motif transitions and the facilitation of their diversification.


Assuntos
Dedos de Zinco CYS2-HIS2 , DNA/metabolismo , Evolução Molecular , Animais , Humanos , Linhagem , Ligação Proteica
8.
Annu Rev Pharmacol Toxicol ; 57: 39-60, 2017 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-27618737

RESUMO

Protein-protein interactions are fundamental for virtually all functions of the cell. A large fraction of these interactions involve short peptide motifs, and there has been increased interest in targeting them using peptide-based therapeutics. Peptides benefit from being specific, relatively safe, and easy to produce. They are also easy to modify using chemical synthesis and molecular biology techniques. However, significant challenges remain regarding the use of peptides as therapeutic agents. Identification of peptide motifs is difficult, and peptides typically display low cell permeability and sensitivity to enzymatic degradation. In this review, we outline the principal high-throughput methodologies for motif discovery and describe current methods for overcoming pharmacokinetic and bioavailability limitations.


Assuntos
Descoberta de Drogas/métodos , Biblioteca de Peptídeos , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Animais , Descoberta de Drogas/tendências , Humanos , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia
9.
Nucleic Acids Res ; 43(19): 9147-57, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26384429

RESUMO

Development of an accurate protein-DNA recognition code that can predict DNA specificity from protein sequence is a central problem in biology. C2H2 zinc fingers constitute by far the largest family of DNA binding domains and their binding specificity has been studied intensively. However, despite decades of research, accurate prediction of DNA specificity remains elusive. A major obstacle is thought to be the inability of current methods to account for the influence of neighbouring domains. Here we show that this problem can be addressed using a structural approach: we build structural models for all C2H2-ZF-DNA complexes with known binding motifs and find six distinct binding modes. Each mode changes the orientation of specificity residues with respect to the DNA, thereby modulating base preference. Most importantly, the structural analysis shows that residues at the domain interface strongly and predictably influence the binding mode, and hence specificity. Accounting for predicted binding mode significantly improves prediction accuracy of predicted motifs. This new insight into the fundamental behaviour of C2H2-ZFs has implications for both improving the prediction of natural zinc finger-binding sites, and for prioritizing further experiments to complete the code. It also provides a new design feature for zinc finger engineering.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Dedos de Zinco , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Bases de Dados de Proteínas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Análise de Sequência de Proteína
10.
Nat Biotechnol ; 33(5): 555-62, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25690854

RESUMO

Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors. However, for most C2H2-ZF proteins it is unknown whether they even bind DNA or, if they do, to which sequences. Here, by combining data from a modified bacterial one-hybrid system with protein-binding microarray and chromatin immunoprecipitation analyses, we show that natural C2H2-ZFs encoded in the human genome bind DNA both in vitro and in vivo, and we infer the DNA recognition code using DNA-binding data for thousands of natural C2H2-ZF domains. In vivo binding data are generally consistent with our recognition code and indicate that C2H2-ZF proteins recognize more motifs than all other human transcription factors combined. We provide direct evidence that most KRAB-containing C2H2-ZF proteins bind specific endogenous retroelements (EREs), ranging from currently active to ancient families. The majority of C2H2-ZF proteins, including KRAB proteins, also show widespread binding to regulatory regions, indicating that the human genome contains an extensive and largely unstudied adaptive C2H2-ZF regulatory network that targets a diverse range of genes and pathways.


Assuntos
Proteínas de Transporte/metabolismo , Genoma Humano , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Retroelementos/genética , Proteínas de Transporte/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/genética , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética
11.
J Mol Biol ; 425(16): 2910-21, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23702294

RESUMO

Eukaryotic chromosomes are capped by telomeres, nucleoprotein complexes that prevent chromosome end-to-end fusions and control cell ageing. Two proteins in this complex, telomere repeat binding factors (TRF1 and TRF2), specifically recognise the double-stranded TTAGGG tandem repeat sequence. TRF1 is a homodimer with roles governing DNA architecture and negatively regulating telomere length. We explore the conformational space of this protein-DNA complex using molecular dynamics and, for the first time, generate a complete model of TRF1-DNA recognition that has not been possible on the basis of crystallographic and NMR data alone. The results reconcile previous conflicting experimental models for the sequence selectivity of the recognition process, by confirming many of the findings while identifying important new interactions and behaviour. This improved characterisation also reveals extensive indirect readout, which suggests that recognition will be affected by changes to DNA helical parameters such as bending.


Assuntos
Telômero/química , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica
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