Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.

• Klíčová slova
• PIN1, dimerization, hydrophilic hoop, intrinsic disorder, subcellular trafficking,
• MeSH
• Arabidopsis * metabolismus   MeSH
• biologický transport MeSH
• kořeny rostlin metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• vnitřně neuspořádané proteiny * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku * MeSH
• vnitřně neuspořádané proteiny * MeSH

Density functional theory was employed to study the influence of O-phosphorylation of serine, threonine, and tyrosine on the amidic (15)N chemical shielding anisotropy (CSA) tensor in the context of the complex chemical environments of protein structures. Our results indicate that the amidic (15)N CSA tensor has sensitive responses to the introduction of the phosphate group and the phosphorylation-promoted rearrangement of solvent molecules and hydrogen bonding networks in the vicinity of the phosphorylated site. Yet, the calculated (15)N CSA tensors in phosphorylated model peptides were in range of values experimentally observed for non-phosphorylated proteins. The extent of the phosphorylation induced changes suggests that the amidic (15)N CSA tensor in phosphorylated proteins could be reasonably well approximated with averaged CSA tensor values experimentally determined for non-phosphorylated amino acids in practical NMR applications, where chemical surrounding of the phosphorylated site is not known a priori in majority of cases. Our calculations provide estimates of relative errors to be associated with the averaged CSA tensor values in interpretations of NMR data from phosphorylated proteins.

• MeSH
• anizotropie MeSH
• fosfáty chemie   MeSH
• fosforylace MeSH
• izotopy dusíku chemie   MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• peptidy chemie   MeSH
• proteiny chemie   MeSH
• rozpouštědla chemie   MeSH
• serin chemie   MeSH
• threonin chemie   MeSH
• tyrosin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfáty MeSH
• izotopy dusíku MeSH
• peptidy MeSH
• proteiny MeSH
• rozpouštědla MeSH
• serin MeSH
• threonin MeSH
• tyrosin MeSH

Cyclin-dependent kinase 2 (CDK2) is the most thoroughly studied of the cyclin-dependent kinases that regulate essential cellular processes, including the cell cycle, and it has become a model for studies of regulatory mechanisms at the molecular level. This contribution identifies flexible and rigid regions of CDK2 based on temperature B-factors acquired from both X-ray data and molecular dynamics simulations. In addition, the biological relevance of the identified flexible regions and their motions is explored using information from the essential dynamics analysis related to conformational changes of CDK2 and knowledge of its biological function(s). The conserved regions of CMGC protein kinases' primary sequences are located in the most rigid regions identified in our analyses, with the sole exception of the absolutely conserved G13 in the tip of the glycine-rich loop. The conserved rigid regions are important for nucleotide binding, catalysis, and substrate recognition. In contrast, the most flexible regions correlate with those where large conformational changes occur during CDK2 regulation processes. The rigid regions flank and form a rigid skeleton for the flexible regions, which appear to provide the plasticity required for CDK2 regulation. Unlike the rigid regions (which as mentioned are highly conserved) no evidence of evolutionary conservation was found for the flexible regions.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• cyklin-dependentní kinasa 2 chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• molekulární modely MeSH
• sekvence aminokyselin MeSH
• termodynamika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• CDK2 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 MeSH

Molecular dynamics (MD) simulations were used to explain structural details of cyclin-dependent kinase-2 (CDK2) inhibition by phosphorylation at T14 and/or Y15 located in the glycine-rich loop (G-loop). Ten-nanosecond-long simulations of fully active CDK2 in a complex with a short peptide (HHASPRK) substrate and of CDK2 inhibited by phosphorylation of T14 and/or Y15 were produced. The inhibitory phosphorylations at T14 and/or Y15 show namely an ATP misalignment and a G-loop shift (~5 A) causing the opening of the substrate binding box. The biological functions of the G-loop and GxGxxG motif evolutionary conservation in protein kinases are discussed. The position of the ATP gamma-phosphate relative to the phosphorylation site (S/T) of the peptide substrate in the active CDK2 is described and compared with inhibited forms of CDK2. The MD results clearly provide an explanation previously not known as to why a basic residue (R/K) is preferred at the P(2) position in phosphorylated S/T peptide substrates.

• MeSH
• adenosintrifosfát chemie   MeSH
• aminokyselinové motivy MeSH
• časové faktory MeSH
• cyklin-dependentní kinasa 2 MeSH
• fosfáty chemie   MeSH
• fosforylace MeSH
• hořčík chemie   MeSH
• inhibitory enzymů chemie   MeSH
• ionty MeSH
• kinasy CDC2-CDC28 antagonisté a inhibitory   chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• peptidy chemie   MeSH
• rentgenové záření MeSH
• software MeSH
• stereoizomerie MeSH
• terciární struktura proteinů MeSH
• threonin chemie   MeSH
• tyrosin chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• CDK2 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 MeSH
• fosfáty MeSH
• hořčík MeSH
• inhibitory enzymů MeSH
• ionty MeSH
• kinasy CDC2-CDC28 MeSH
• peptidy MeSH
• threonin MeSH
• tyrosin MeSH

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.

• MeSH
• aktivace enzymů MeSH
• cyklin-dependentní kinasa 2 MeSH
• fosforylace MeSH
• fosfotyrosin metabolismus   MeSH
• glycin metabolismus   MeSH
• kinasy CDC2-CDC28 antagonisté a inhibitory   chemie   metabolismus   MeSH
• molekulární modely MeSH
• sekundární struktura proteinů MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasa 2 MeSH
• fosfotyrosin MeSH
• glycin MeSH
• kinasy CDC2-CDC28 MeSH