Domain Interactions on the ntr Signal Transduction Pathway: Two-Hybrid Analysis of Mutant and Truncated Derivatives of Histidine Kinase NtrB

We have used the yeast two-hybrid system to analyze protein-protein interactions mediated by domains of regulatory proteins of the ntr signal transduction system, including interactions among NtrB derivatives and their interactions with NtrC and PII from Klebsiella pneumoniae. Interactions took place only between proteins or protein domains belonging to the ntr signal transduction system and not between proteins or domains from noncognate regulators. NtrB and its transmitter domain, but not NtrC, CheA, or the cytoplasmic C terminus of EnvZ, interacted with PII. In addition, interaction of NtrB with NtrC, but not with PII, depended on the histidine phosphotransfer domain. Point mutation A129T, diminishing the NtrC phosphatase activity of NtrB, affected the strength of the signals between NtrC and the transmitter module of NtrB but had no impact on PII signals, suggesting that A129T prevents the conformational change needed by NtrB to function as a phosphatase for NtrC, rather than disturbing binding to PII.     

Accelerated response of the myogenin gene to denervation in mutant mice lacking phosphorylation of myogenin at threonine 87

Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation.     

Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF₃ ⁻ addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.     

Conformational switching upon phosphorylation: a predictive framework based on energy landscape principles

We investigate how post-translational phosphorylation modifies the global conformation of a protein by changing its free energy landscape using two test proteins, cystatin and NtrC. We first examine the changes in a free energy landscape caused by phosphorylation using a model containing information about both structural forms. For cystatin the free energy cost is fairly large indicating a low probability of sampling the phosphorylated conformation in a perfectly funneled landscape. The predicted barrier for NtrC conformational transition is several times larger than the barrier for cystatin, indicating that the switch protein NtrC most probably follows a partial unfolding mechanism to move from one basin to the other. Principal component analysis and linear response theory show how the naturally occurring conformational changes in unmodified proteins are captured and stabilized by the change of interaction potential. We also develop a partially guided structure prediction Hamiltonian which is capable of predicting the global structure of a phosphorylated protein using only knowledge of the structure of the unphosphorylated protein or vice versa. This algorithm makes use of a generic transferable long-range residue contact potential along with details of structure short range in sequence. By comparing the results obtained with this guided transferable potential to those from the native-only, perfectly funneled Hamiltonians, we show that the transferable Hamiltonian correctly captures the nature of the global conformational changes induced by phosphorylation and can sample substantially correct structures for the modified protein with high probability.     

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.     

Defective protein histidine phosphorylation in islets from the Goto-Kakizaki diabetic rat

We recently described novel regulatory roles for protein histidine phosphorylation of key islet proteins (e.g., nucleoside diphosphate kinase and succinyl thiokinase) in insulin secretion from the islet beta-cell (Kowluru A. Diabetologia 44: 89-94, 2001; Kowluru A, Tannous M, and Chen HQ. Arch Biochem Biophys 398: 160-169, 2002). In this context, we also characterized a novel, ATP- and GTP-sensitive protein histidine kinase in isolated beta-cells that catalyzed the histidine phosphorylation of islet (endogenous) proteins as well as exogenously added histone 4, and we implicated this kinase in the activation of islet endogenous G proteins (Kowluru A. Biochem Pharmacol 63: 2091-2100, 2002). In the present study, we describe abnormalities in ATP- or GTP-mediated histidine phosphorylation of nucleoside diphosphate kinase in islets derived from the Goto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes. Furthermore, we provide evidence for a marked reduction in the activities of ATP- or GTP-sensitive histidine kinases in GK rat islets. On the basis of these observations, we propose that alterations in protein histidine phosphorylation could contribute toward insulin-secretory abnormalities demonstrable in the diabetic islet.     

Coarse-grained dynamics of the receiver domain of NtrC: fluctuations, correlations and implications for allosteric cooperativity

Receiver domains are key molecular switches in bacterial signaling. Structural studies have shown that the receiver domain of the nitrogen regulatory protein C (NtrC) exists in a conformational equilibrium encompassing both inactive and active states, with phosphorylation of Asp54 allosterically shifting the equilibrium towards the active state. To analyze dynamical fluctuations and correlations in NtrC as it undergoes activation, we have applied a coarse-grained dynamics algorithm using elastic network models. Normal mode analysis reveals possible dynamical pathways for the transition of NtrC from the inactive state to the active state. The diagonalized correlation between the inactive and the active (phosphorylated) state shows that most correlated motions occur around the active site of Asp54 and in the region Thr82 to Tyr101. This indicates a coupled correlation of dynamics in the "Thr82-Tyr101" motion. With phosphorylation inducing significant flexibility changes around the active site and alpha3 and alpha4 helices, we find that this activation makes the active-site region and the loops of alpha3/beta4 and alpha4/beta5 more stable. This means that phosphorylation entropically favors the receiver domain in its active state, and the induced conformational changes occur in an allosteric manner. Analyses of the local flexibility and long-range correlated motion also suggest a dynamics criterion for determining the allosteric cooperativity of NtrC, and may be applicable to other proteins.