Structural bases of unphosphorylated STAT1 association and receptor binding

The crystal structure has been determined at 3.0 A resolution for an unphosphorylated STAT1 (1-683) complexed with a phosphopeptide derived from the alpha chain of interferon gamma (IFNgamma) receptor. Two dimer interfaces are seen, one between the N domains (NDs) (amino acid residues 1-123) and the other between the core fragments (CFs) (residues 132-683). Analyses of the wild-type (wt) and mutant STAT1 proteins by static light scattering, analytical ultracentrifugation, and coimmunoprecipitation suggest that STAT1 is predominantly dimeric prior to activation, and the dimer is mediated by the ND interactions. The connecting region between the ND and the CF is flexible and allows two interconvertable orientations of the CFs, termed "antiparallel" or "parallel," as determined by SH2 domain orientations. Functional implications of these dimer conformations are discussed. Also revealed in this structure is the detailed interaction between STAT1 SH2 domain and its docking site on IFNgamma receptor.     

Quaternary structure and function in phage lambda repressor: 1H-NMR studies of genetically altered proteins

The quaternary structure and dynamics of phage lambda repressor are investigated in solution by 1H-NMR methods. lambda repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. 1H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the "linker" polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84----Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.     

Quarternary Structure and Function in Phage λ Repressor: 1H-NMR Studies of Genetically Altered Proteins

Abstract

The quaternary structure and dynamics of phage λ repressor are investigated in solution by ¹H-NMR methods. λ repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. ¹H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the “linker” polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84→Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.     

Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein.

Retroviral integrase, an essential enzyme for replication of human immunodeficiency virus type-1 (HIV-1) and other retroviruses, contains three structurally distinct domains, an N-terminal domain, the catalytic core and a C-terminal domain. To elucidate their spatial arrangement, we have solved the structure of a fragment of HIV-1 integrase comprising the N-terminal and catalytic core domains. This structure reveals a dimer interface between the N-terminal domains different from that observed for the isolated domain. It also complements the previously determined structure of the C-terminal two domains of HIV-1 integrase; superposition of the conserved catalytic core of the two structures results in a plausible full-length integrase dimer. Furthermore, an integrase tetramer formed by crystal lattice contacts bears structural resemblance to a related bacterial transposase, Tn5, and exhibits positively charged channels suitable for DNA binding.     

Evidence for phosphorylation-dependent conformational changes in methylesterase CheB

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N-terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N-terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.     

Dimer-induced signal propagation in Spo0A

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.     

The dimeric form of the unphosphorylated response regulator BaeR

Bacterial response regulators (RRs) can regulate the expression of genes that confer antibiotic resistance; they contain a receiver and an effector domain and their ability to bind DNA is based on the dimerization state. This is triggered by phosphorylation of the receiver domain by a kinase. However, even in the absence of phosphorylation RRs can exist in equilibrium between monomers and dimers with phosphorylation shifting the equilibrium toward the dimer form. We have determined the crystal structure of the unphosphorylated dimeric BaeR from Escherichia coli. The dimer interface is formed by a domain swap at the receiver domain. In comparison with the unphosphorylated dimeric PhoP from Mycobacterium tuberculosis, BaeR displays an asymmetry of the effector domains.     

Tyr-317 phosphorylation increases Shc structural rigidity and reduces coupling of domain motions remote from the phosphorylation site as revealed by molecular dynamics simulations

Activated receptor tyrosine kinases bind the Shc adaptor protein through its N-terminal phosphotyrosine-binding (PTB) and C-terminal Src homology 2 (SH2) domains. After binding, Shc is phosphorylated within the central collagen-homology (CH) linker region on Tyr-317, a residue remote to both the PTB and SH2 domains. Shc phosphorylation plays a pivotal role in the initiation of mitogenic signaling through the Ras/Raf/MEK/ERK pathway, but it is unclear if Tyr-317 phosphorylation affects Shc-receptor interactions through the PTB and SH2 domains. To investigate the structural impact of Shc phosphorylation, molecular dynamics simulations were carried out using special-purpose Molecular Dynamics Machine-Grape computers. After a 1-nanosecond equilibration, atomic motions in the structures of unphosphorylated Shc and Shc phosphorylated on Tyr-317 were calculated during a 2-nanosecond period. The results reveal larger phosphotyrosine-binding domain fluctuations and more structural flexibility of unphosphorylated Shc compared with phosphorylated Shc. Collective motions between the PTB-SH2, PTB-CH, and CH-SH2 domains were highly correlated only in unphosphorylated Shc. Dramatic changes in domain coupling and structural rigidity, induced by Tyr-317 phosphorylation, may alter Shc function, bringing about marked differences in the association of unphosphorylated and phosphorylated Shc with its numerous partners, including activated membrane receptors.     

A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules.

During signal transduction, response regulators of two-component systems are phosphorylated in a conserved receiver module. Phosphorylation induces activation of the non-conserved output domain. We fused various domains of the response regulators NtrC, PhoB or CheB to the DNA binding domain of lambda repressor. Analysis of these hybrid proteins shows that the receiver modules of NtrC and PhoB are potential dimerization domains. In the unphosphorylated proteins, the ability of the receiver modules to dimerize is masked due to inhibition by their output domains. Inhibition can be relieved in two ways: phosphorylation of the receiver module or deletion of the output domain. In contrast, the receiver module of CheB lacks this ability for dimerization. We propose a model which groups response regulators into two classes. Common to both classes is the interaction between receiver and output domain in the unphosphorylated protein. In class I (e.g. NtrC and PhoB), this interaction leads to the inhibition of the receiver module. Phosphorylation relieves inhibition, thereby inducing activation via dimerization of the receiver modules. In class II (e.g. CheB), the interaction between receiver and output domain results in inhibition of the output domain. Phosphorylation relieves inhibition, thereby activating the output domain.     

Structure of the response regulator PhoP from Mycobacterium tuberculosis reveals a dimer through the receiver domain

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving α4-β5-α5, a common interface for activated receiver domain dimers. However, the switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.     

Molluscan catch muscle myorod and its N-terminal peptide bind to F-actin and myosin in a phosphorylation-dependent manner

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. This protein is an alternatively spliced product of the myosin heavy-chain gene containing the C-terminal rod part of myosin and a unique N-terminal domain. We have recently reported that this unique domain is a target for phosphorylation by gizzard smooth muscle myosin light chain kinase (MLCK) and molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod phosphorylation in catch muscle, a peptide corresponding to the specific N-terminal region of the protein was synthesized in phosphorylated and unphosphorylated form. We report, for the first time, that unphosphorylated full-length myorod and its unphosphorylated N-terminal synthetic peptide are able to interact with rabbit F-actin and thin filaments from molluscan catch muscle. The binding between thin filaments and the peptide was Ca²⁺-dependent. In addition, we found that phosphorylated N-terminal peptide of myorod has higher affinity for myosin compared to the unphosphorylated peptide. Together, these observations suggest the direct involvement of the N-terminal domain of myorod in the regulation of molluscan catch muscle.     

Stability and binding of the phosphorylated species of the N-terminal domain of enzyme I and the histidine phosphocarrier protein from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system

The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. It is formed by two general proteins: enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI is formed by two domains, with the N-terminal domain (EIN) being responsible for the binding to HPr. In low-G+C Gram-positive bacteria, HPr becomes phosphorylated not only by phosphoenolpyruvate (PEP) at the active-site histidine, but also by ATP at a serine. In this work, we have characterized: (i) the stability and binding affinities between the active-site-histidine phosphorylated species of HPr and the EIN from Streptomyces coelicolor; and (ii) the stability and binding affinities of the species involving the phosphorylation at the regulatory serine of HPr(sc). Our results show that the phosphorylated active-site species of both proteins are less stable than the unphosphorylated counterparts. Conversely, the Hpr-S47D, which mimics phosphorylation at the regulatory serine, is more stable than wild-type HPr(sc) due to helical N-capping effects, as suggested by the modeled structure of the protein. Binding among the phosphorylated and unphosphorylated species is always entropically driven, but the affinity and the enthalpy vary widely.     

Structural and functional studies of the response regulator HupR

HupR is a response regulator that controls the synthesis of the membrane-bound [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus. The protein belongs to the NtrC subfamily of response regulators and is the second protein of a two-component system. We have crystallized the full-length protein HupR in the unphosphorylated state in two dimensions using the lipid monolayer technique. The 3D structure of negatively stained HupR was calculated to a resolution of approximately 23 A from tilted electron microscope images. HupR crystallizes as a dimer, and forms an elongated V-shaped structure with extended arms. The dimensions of the dimer are about 80 A length, 40 A width and 85 A thick. The HupR monomer consists of three domains, N-terminal receiver domain, central domain and C-terminal DNA-binding domain. We have fitted the known 3D structure of the central domain from NtrC1 Aquifex aeolicus protein into our 3D model; we propose that contact between the dimers is through the central domain. The N-terminal domain is in contact with the lipid monolayer and is situated on the top of the V-shaped structure. The central domain alone has been expressed and purified; it forms a pentamer in solution and lacks ATPase activity.