RESUMO
Cyclin-dependent kinases (CDKs), in association with cyclins, control cell cycle progression by phosphorylating a large number of substrates. In animals, activation of CDKs regularly requires both the association with a cyclin and then phosphorylation of a highly conserved threonine residue in the CDK activation loop (the classical mechanism), mediated by a CDK-activating kinase (CAK). In addition to this typical mechanism of activation, some CDKs can also be activated by the association of a cyclin to a monomeric CDK previously phosphorylated by CAK although not all CDKs can be activated by this mechanism. In animals and yeast, cyclin, in addition to being required for CDK activation, provides substrate specificity to the cyclin/CDK complex; however, in plants both the mechanisms of CDKs activation and the relevance of the CDK-associated cyclin for substrate targeting have been poorly studied. In this work, by co-expressing proteins in E. coli, we studied maize CDKA2;1a and CDKB1;1, two of the main types of CDKs that control the cell cycle in plants. These kinases could be activated by the classical mechanism and by the association of CycD2;2a to a phosphorylated intermediate in its activation loop, a previously unproven mechanism for the activation of plant CDKs. Unlike CDKA2;1a, CDKB1;1 did not require CAK for its activation, since it autophosphorylated in its activation loop. Phosphorylation of CDKB1;1 and association of CycD2;2 was not enough for its full activation as association of maize CKS, a scaffolding protein, differentially stimulated substrate phosphorylation. Our results suggest that both CDKs participate in substrate recognition.
Assuntos
Proteínas Serina-Treonina Quinases , Zea mays , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Zea mays/genética , Escherichia coli/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Methanogenic archaea have received attention due to their potential use in biotechnological applications such as methane production, so their metabolism and regulation are topics of special interest. When growing in a nutrient-rich medium, these organisms exhibit gluconeogenic metabolism; however, under starvation conditions, they turn to glycolytic metabolism. To date, no regulatory mechanism has been described for this gluconeogenic/glycolytic metabolic switch. Here, we report that adenosine monophosphate (AMP) activates both enzymatic activities of the bifunctional adenosine diphosphate (ADP)-dependent phosphofructokinase/glucokinase from Methanococcus maripaludis (MmPFK/GK). To understand this phenomenon, we performed a comprehensive kinetic characterisation, including determination of the kinetics, substrate inhibition and AMP activation mechanism of this enzyme. We determined that MmPFK/GK has an ordered-sequential mechanism, in which MgADP is the first substrate to bind and AMP is the last product released. The enzyme also displays substrate inhibition by both sugar substrates; we determined that this inhibition occurs through the formation of catalytically nonproductive enzyme complexes caused by sugar binding. For both activities, the AMP activation mechanism occurs primarily through incremental changes in the affinity for the sugar substrate, with this effect being higher in the GK than in the PFK activity. Interestingly, due to the increase in the sugar substrate affinity caused by AMP, an enhancement in the sugar substrate inhibition effect was also observed for both activities, which can be explained by an increase in sugar binding leading to the formation of dead-end complexes. These results shed light on the regulatory mechanisms of methanogenic archaeal sugar metabolism, a phenomenon that has been largely unexplored.
Assuntos
Mathanococcus , Fosfofrutoquinases , Difosfato de Adenosina , Monofosfato de Adenosina , Mathanococcus/genética , AçúcaresRESUMO
The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain.IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Brucella abortus/metabolismo , Cor , Histidina Quinase/química , Histidina Quinase/metabolismo , Luz , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/patogenicidade , Histidina Quinase/genética , Modelos Moleculares , Domínios Proteicos , Transdução de SinaisRESUMO
Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.