RESUMO
Data from gnomAD indicate that a missense mutation encoding the T118M variation in human peripheral myelin protein 22 (PMP22) is found in roughly one of every 75 genomes of western European lineage (1:120 in the overall human population). It is unusual among PMP22 variants that cause Charcot-Marie-Tooth (CMT) disease in that it is not 100% penetrant. Here, we conducted cellular and biophysical studies to determine why T118M PMP22 predisposes humans to CMT, but with only incomplete penetrance. We found that T118M PMP22 is prone to mistraffic but differs even from the WT protein in that increased expression levels do not result in a reduction in trafficking efficiency. Moreover, the T118M mutant exhibits a reduced tendency to form large intracellular aggregates relative to other disease mutants and even WT PMP22. NMR spectroscopy revealed that the structure and dynamics of T118M PMP22 resembled those of WT. These results show that the main consequence of T118M PMP22 in WT/T118M heterozygous individuals is a reduction in surface-trafficked PMP22, unaccompanied by formation of toxic intracellular aggregates. This explains the incomplete disease penetrance and the mild neuropathy observed for WT/T118M CMT cases. We also analyzed BioVU, a biobank linked to deidentified electronic medical records, and found a statistically robust association of the T118M mutation with the occurrence of long and/or repeated episodes of carpal tunnel syndrome. Collectively, our results illuminate the cellular effects of the T118M PMP22 variation leading to CMT disease and indicate a second disorder for which it is a risk factor.
Assuntos
Doença de Charcot-Marie-Tooth , Proteínas da Mielina , Humanos , Doença de Charcot-Marie-Tooth/genética , Mutação de Sentido Incorreto , Proteínas da Mielina/genética , Predisposição Genética para DoençaRESUMO
Peripheral myelin protein (PMP22) is an integral membrane protein that traffics inefficiently even in wild-type (WT) form, with only 20% of the WT protein reaching its final plasma membrane destination in myelinating Schwann cells. Misfolding of PMP22 has been identified as a key factor in multiple peripheral neuropathies, including Charcot-Marie-Tooth disease and Dejerine-Sottas syndrome. While biophysical analyses of disease-associated PMP22 mutants show altered protein stabilities, leading to reduced surface trafficking and loss of PMP22 function, it remains unclear how destabilization of PMP22 mutations causes mistrafficking. Here, native ion mobility-mass spectrometry (IM-MS) is used to compare the gas phase stabilities and abundances for an array of mutant PM22 complexes. We find key differences in the PMP22 mutant stabilities and propensities to form homodimeric complexes. Of particular note, we observe that severely destabilized forms of PMP22 exhibit a higher propensity to dimerize than WT PMP22. Furthermore, we employ lipid raft-mimicking SCOR bicelles to study PMP22 mutants, and find that the differences in dimer abundances are amplified in this medium when compared to micelle-based data, with disease mutants exhibiting up to 4 times more dimer than WT when liberated from SCOR bicelles. We combine our findings with previous cellular data to propose that the formation of PMP22 dimers from destabilized monomers is a key element of PMP22 mistrafficking.
Assuntos
Proteínas da Mielina/metabolismo , Doenças do Sistema Nervoso Periférico/fisiopatologia , Transporte Proteico/fisiologia , Membrana Celular/metabolismo , Humanos , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Proteínas da Mielina/genética , Proteínas da Mielina/fisiologia , Doenças do Sistema Nervoso Periférico/diagnóstico por imagem , Doenças do Sistema Nervoso Periférico/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Células de Schwann/metabolismoRESUMO
There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-ß-melibioside (ß-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1â6)-d-glucose. Light scattering showed the ß-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-ß-maltoside (ß-DDM). ß-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into ß-DDMB yielded activities that were 40% higher than those of DAGK purified into ß-DDM. ß-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. ß-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Detergentes/química , Diacilglicerol Quinase/metabolismo , Dissacarídeos/química , Proteínas de Escherichia coli/metabolismo , Glicolipídeos/química , Proteínas da Mielina/metabolismo , Receptor Notch1/metabolismo , Precursor de Proteína beta-Amiloide/química , Detergentes/síntese química , Diacilglicerol Quinase/química , Dissacarídeos/síntese química , Difusão Dinâmica da Luz , Estabilidade Enzimática , Proteínas de Escherichia coli/química , Glucosídeos/química , Glicolipídeos/síntese química , Temperatura Alta/efeitos adversos , Humanos , Micelas , Proteínas da Mielina/química , Ressonância Magnética Nuclear Biomolecular , Tamanho da Partícula , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Receptor Notch1/químicaRESUMO
Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme's conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5-24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme's fold.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Difosfato de Adenosina/metabolismo , Animais , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Deutério/análise , Hidrogênio/análise , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação ProteicaRESUMO
NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR-especially human membrane proteins-and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.
RESUMO
ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.
RESUMO
Recent advances in whole genome and exome sequencing have dramatically increased the database of human gene variations. There are now enough sequenced human exomes and genomes to begin to identify gene variations that are notable because they are NOT observed in sequenced human genomes, apparently because they are subject to "purifying selection", exemplifying genetic intolerance. Such "dysprocreative" gene variations are embryonic lethal or prevent reproduction through any one of a number of possible mechanisms. Here we review an emerging quantitative approach, "Missense Tolerance Ratio" (MTR) analysis, that is used to assess protein-encoding gene (cDNA) sequence intolerance to missense mutations based on analysis of the >100â¯K and growing number of currently available human genome and exome sequences. This approach is already useful for analyzing intolerance to mutations in cDNA segments with a resolution on the order of 90 bases. Moreover, as the number of sequenced genomes/exomes increases by orders of magnitude it may eventually be possible to assess mutational tolerance in a statistically robust manner at or near single site resolution. Here we focus on how cDNA intolerance analysis complements other bioinformatic methods to illuminate structure-folding-function relationships for the encoded proteins. A set of disease-linked membrane proteins is employed to provide examples.
Assuntos
Biologia Computacional/métodos , Exoma/genética , Mutação de Sentido Incorreto , Análise de Sequência de DNA , Animais , Variação Genética/genética , Genoma Humano/genética , Humanos , Mutação de Sentido Incorreto/genética , Dobramento de ProteínaRESUMO
In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.