RESUMO
Adenylate kinase, an enzyme that catalyzes the phosphoryl transfer between ATP and AMP, can interconvert between the open and catalytically potent (closed) forms even without binding ligands. Several aspects of the enzyme elasticity and internal dynamics are analyzed here by atomistic molecular dynamics simulations covering a total time span of 100 ns. This duration is sufficiently long to reveal a partial conversion of the enzyme that proceeds through jumps between structurally different substates. The intra- and intersubstates contributions to the enzyme's structural fluctuations are analyzed and compared both in magnitude and directionality. It is found that, despite the structural heterogeneity of the visited conformers, the generalized directions accounting for conformational fluctuations within and across the substates are mutually consistent and can be described by a limited set of collective modes. The functional-oriented nature of the consensus modes is suggested by their good overlap with the deformation vector bridging the open and closed crystal structures. The consistency of adenylate kinase's internal dynamics over timescales wide enough to capture intra- and intersubstates fluctuations adds elements in favor of the recent proposal that the free (apo) enzyme possesses an innate ability to sustain the open/close conformational changes.
Assuntos
Adenilato Quinase/química , Adenilato Quinase/metabolismo , Escherichia coli/enzimologia , Movimento , Fenômenos Biomecânicos , Estrutura Terciária de ProteínaRESUMO
Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here, we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using nuclear magnetic resonance (NMR) spectroscopy, stopped-flow kinetics, and isothermal titration calorimetry, we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding.
Assuntos
Receptor Quinase 1 Acoplada a Proteína G/química , Recoverina/química , Escherichia coli , Receptor Quinase 1 Acoplada a Proteína G/genética , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Quaternária de Proteína , Recoverina/genética , Recoverina/metabolismoRESUMO
Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg(2+) cofactor activates two distinct molecular events: phosphoryl transfer (>10(5)-fold) and lid opening (10(3)-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 10(3)-fold without substantially affecting lid opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.
Assuntos
Adenilato Quinase/química , Adenilato Quinase/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos MolecularesRESUMO
The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the "Y-T coupling" mechanism, wherein the χ1 rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrC(R)), the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to χ1 rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrC(R) in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provide experimental evidence against the generally accepted "Y-T coupling" mechanism of activation in NtrC(R).
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Sítio Alostérico , Simulação por Computador , Proteínas PII Reguladoras de Nitrogênio/metabolismoRESUMO
We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX2(1-25). While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases.DOI: http://dx.doi.org/10.7554/eLife.02667.001.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Regulação Alostérica , Aurora Quinase A/química , Biocatálise , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Humanos , Cinética , Proteínas Associadas aos Microtúbulos/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Soluções , Especificidade por SubstratoRESUMO
Low energy modes have been calculated for the largest possible number of available representatives (>150) of EF-hand domains belonging to different members of the calcium-binding EF-hand protein superfamily. These proteins are the major actors in signal transduction. The latter, in turn, relies on the dynamical properties of the systems, in particular on the relative movements of the four helices characterizing each EF-hand domain upon calcium binding. The peculiar structural and dynamical features of this protein superfamily are systematically investigated by a novel approach, where the lowest energy (essential) modes are described in the space of the six interhelical angles among the four helices constituting the EF-hand domain. The modes, obtained through a general and transferable coarse-graining scheme, identify the easy directions of helical motions. It is found that, for most proteins, the two lowest energy modes are sufficient to capture most of the helices' fluctuation dynamics. Strikingly, the comparison of such modes for all possible pairs of EF-hand domain representatives reveals that only few easy directions are preferred within this large protein superfamily. This enables us to introduce a novel dynamics-based classification of EF-hand domains that complements existing structure-based characterizations from an unexplored biological perspective.