Regulation of Raf through phosphorylation and N terminus-C terminus interaction

Raf kinase is a key component in regulating the MAPK pathway. B-Raf has been reported as an oncogene and is mutated in 60% of human melanomas. The main focus of Raf regulation studies has been on phosphorylation, dephosphorylation, and scaffolding proteins; however, Raf also has its own auto-regulatory domain. Removal of the N-terminal regulatory domain, initially discovered in the viral Raf oncogene (v-Raf), results in a kinase domain with high basal activity independent of Ras activation. In this report, we show that activating phosphorylations are still required for activity of the truncated C-terminal kinase domain (called 22W). The interaction between the N-terminal regulatory domain and the C-terminal kinase domain is disrupted by activated Ras. Mutations in the Ras binding domain, cysteine-rich domain, or S259A do not affect the inhibition of 22W by the N-terminal domain. When phosphomimetic residues are substituted at the activating sites (DDED) in 22W, this results in a higher basal activity that is no longer inhibited by expression of the N-terminal domain, although binding to the N-terminal domain still occurs. Although the interaction between 22W/DDED and the N-terminal domain may be in a different conformation, the interaction is still disrupted by activated Ras. These data demonstrate that N-terminal domain binding to the kinase domain inhibits the activity of the kinase domain. However, this inhibition is relieved when the C-terminal kinase domain is activated by phosphorylation.     

The cooperative role of OsCnfU-1A domain I and domain II in the iron sulphur cluster transfer process as revealed by NMR

OsCnfU-1A is a chloroplast-type Nfu-like protein that consists of tandem repeats sharing high sequence homology. Domain I of this protein, but not domain II, has a C-X-X-C motif that is thought to assemble an iron-sulphur cluster. Herein we report the solution structure of OsCnfU-1A domain I (73-153). Although OsCnfU-1A domain I is structurally similar to OsCnfU-1A domain II (154-226), the electrostatic surface potential of the 2 domains differs. Domain I has an acidic surface, whereas that of domain II is predominantly basic. Chemical shift perturbation studies on OsCnfU-1A domain I and domain II with ferredoxin revealed negligible chemical shift changes in domain I, whereas much larger chemical shift changes were observed in domain II. The residues with larger chemical shift changes were located on the basic surface of domain II. Considering that ferredoxin is predominantly negatively charged, we propose the following hypothesis: First, an iron-sulphur cluster is assembled on domain I. Next, domain II interacts with the ferredoxin, thus tethering domain I close to the ferredoxin. Finally, domain I transfers the iron-sulphur cluster to the ferredoxin. Thus, domain II facilitates the efficient transfer of the iron-sulphur cluster from domain I to the ferredoxin.     

Definition of the p53 functional domains necessary for inducing apoptosis

The p53 protein contains several functional domains necessary for inducing cell cycle arrest and apoptosis. The C-terminal basic domain within residues 364-393 and the proline-rich domain within residues 64-91 are required for apoptotic activity. In addition, activation domain 2 within residues 43-63 is necessary for apoptotic activity when the N-terminal activation domain 1 within residues 1-42 is deleted (DeltaAD1) or mutated (AD1(-)). Here we have discovered that an activation domain 2 mutation at residues 53-54 (AD2(-)) abrogates the apoptotic activity but has no significant effect on cell cycle arrest. We have also found that p53-(DeltaAD2), which lacks activation domain 2, is inert in inducing apoptosis. p53-(AD2(-)DeltaBD), which is defective in activation domain 2 and lacks the C-terminal basic domain, p53-(DeltaAD2DeltaBD), which lacks both activation domain 2 and the C-terminal basic domain, and p53-(DeltaPRDDeltaBD), which lacks both the proline-rich domain and the C-terminal basic domain, are also inert in inducing apoptosis. All four mutants are still capable of inducing cell cycle arrest, albeit to a lesser extent than wild-type p53. Interestingly, we have found that deletion of the N-terminal activation domain 1 alleviates the requirement of the C-terminal basic domain for apoptotic activity. Thus, we have generated a small but potent p53-(DeltaAD1DeltaBD) molecule. Furthermore, we have determined that at least two of the three domains (activation domain 1, activation domain 2, and the proline-rich domain), are required for inducing cell cycle arrest. Taken together, our results suggest that activation domain 2 and the proline-rich domain form an activation domain for inducing pro-apoptotic genes or inhibiting anti-apoptotic genes. The C-terminal basic domain is required for maintaining this activation domain competent for transactivation or transrepression.     

Stable interdomain interaction within the cytoplasmic domain of CD45 increases enzyme stability

CD45 is a leukocyte-specific, two domain transmembrane tyrosine phosphatase. Co-purification of a recombinant protein containing the first phosphatase domain of CD45 (6His-D1) with a recombinant protein containing the second phosphatase domain (GST-D2) from E. coli indicated a stable interaction which resulted in increased stability of the active phosphatase domain present in 6His-D1. This interaction was not dependent on the acidic region unique to CD45 domain 2, but was affected by a destabilizing point mutation (Q1180G) in GST-D2. CD45 domain 2 enhanced phosphatase activity of the first domain in the full length cytoplasmic domain protein, whereas a chimeric protein with the SH2 domain of p56(lck) in place of the CD45 C-terminal region did not. Thus the C-terminal domain of CD45 associates with the N-terminal domain and this stabilizes the active phosphatase domain. A single destabilizing point mutation in the second domain is sufficient to attenuate this effect.     

Influence of Ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity

Doublecortin-like protein kinase (DCLK) is a Ser/Thr protein kinase predominantly expressed in brain. DCLK is composed of three functional domains; the N-terminal doublecortin-like (DC) domain, the C-terminal kinase domain and Ser/Pro-rich (SP) domain in between DC and kinase domains. Although the DC domain is known to mediate microtubule association, functional roles of the SP domain and the kinase domain on microtubule association is not known. In this study, we investigated the microtubule-binding activity of zebrafish DCLK (zDCLK) using various deletion mutants and chimeric proteins. The microtubule-binding activity of various mutants of zDCLK was assessed both by immunocytochemical analysis and by biochemical analysis using detergent extraction method. When the kinase domain was removed from zDCLK, the microtubule-binding activity was significantly enhanced. Although the zDCLK(DC + SP) mutant showed a strong microtubule-binding activity, the DC domain alone showed much lower microtubule-binding activity, indicating that the SP domain of zDCLK plays a role in enhancing microtubule-binding activity of the DC domain. These results suggest that both the kinase domain and the SP domain are involved in regulating the microtubule-binding activity of DCLK.     

The PA domain: a protease-associated domain

We have identified a similarity between the apical domain of the human transferrin receptor and several other protein families. This domain is found associated with two different families of peptidases. Therefore, we term it the PA domain for protease-associated domain. The PA domain is found inserted within a loop of the peptidase domain of family M8/M33 zinc peptidases. The PA domain is also found in a vacuolar sorting receptor and a ring finger protein of unknown function that may be a cell surface receptor. The PA domain may mediate substrate determination of peptidases or form protein-protein interactions.     

Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound, Kunitz-type serine protease inhibitor. HAI-1 inhibits serine proteases that have potent pro-hepatocyte growth factor-converting activity, such as the membrane-type serine protease, matriptase. HAI-1 comprises an N-terminal domain, followed by an internal domain, first protease inhibitory domain (Kunitz domain I), low-density lipoprotein receptor A module (LDLRA) domain, and a second Kunitz domain (Kunitz domain II) in the extracellular region. Our aim was to assess the roles of these domains in the inhibition of matriptase. Soluble forms of recombinant rat HAI-1 mutants made up with various combinations of domains were produced, and their inhibitory activities toward the hydrolysis of a chromogenic substrate were analyzed using a soluble recombinant rat matriptase. Kunitz domain I exhibited inhibitory activity against matriptase, but Kunitz domain II did not. The N-terminal domain and Kunitz domain II decreased the association rate between Kunitz domain I and matriptase, whereas the internal domain increased this rate. The LDLRA domain suppressed the dissociation of the Kunitz domain I-matriptase complex. Surprisingly, an HAI-1 mutant lacking the N-terminal domain and Kunitz domain II showed an inhibitor constant of 1.6 pm, and the inhibitory activity was 400 times higher in this HAI-1 mutant than in the mutant with all domains. These findings, together with the known occurrence of an HAI-1 species lacking the N-terminal domain and Kunitz domain II in vivo, suggest that the domain structure of HAI-1 is organized in a way that allows HAI-1 to flexibly control matriptase activity.     

Functional characterization of the CFTR R domain using CFTR/MDR1 hybrid and deletion constructs.

To improve our insight into the structure and function of the CFTR R domain, deletion and hybrid constructs in which different parts of the R domain were deleted or replaced by the MDR1 linker domain, and vice versa, were made. Replacement of the linker domain by the R domain did not result in a decrease and replacement of the CFTR R domain by the linker domain did not result in an increase of maturation efficiency, when compared to the respective wild-type proteins. This indicates that the R domain is not responsible for the high degree of degradation observed for CFTR translation products in the ER, but rather the overall structure or sequences located outside the R domain. Replacing the C-terminal part of the R domain (amino acids 780-830) by the MDR1 linker domain resulted in the appearance of PKA-dependent whole cell chloride currents which were not significantly different from wild-type CFTR currents. This might indicate that the PKA sites present in the linker domain are functional and that not the exact sequence of the C-terminal part of the R domain is important, but rather the presence of PKA sites and the length. Moreover, when this hybrid construct was PKC-stimulated, chloride currents were activated. Although these PKC-induced currents were lower than the PKA-induced ones, this again indicates that the linker domain is functional in this hybrid construct. Taken together, these results suggest that the MDR1 linker domain can substitute for part of the regulatory domain of the CFTR protein.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

Crystal structure of the bovine mitochondrial elongation factor Tu.Ts complex

The three-dimensional structure of the bovine mitochondrial elongation factor (EF)-Tu.Ts complex (EF-Tumt.Tsmt) has been determined to 2.2-A resolution using the multi-wavelength anomalous dispersion experimental method. This complex provides the first insight into the structure of EF-Tsmt. EF-Tsmt is similar to Escherichia coli and Thermus thermophilus EF-Ts in the amino-terminal domain. However, the structure of EF-Tsmt deviates considerably in the core domain with a five-stranded beta-sheet forming a portion of subdomain N of the core. In E. coli EF-Ts, this region is composed of a three-stranded sheet. The coiled-coil domain of the E. coli EF-Ts is largely eroded in EF-Tsmt, in which it consists of a large loop packed against subdomain C of the core. The conformation of bovine EF-Tumt in complex with EF-Tsmt is distinct from its conformation in the EF-Tumt.GDP complex. When domain III of bovine EF-Tumt.GDP is superimposed on domain III of EF-Tumt in the EF-Tumt.Tsmt complex, helix B from domain I is also almost superimposed. However, the rest of domain I is rotated relative to this helix toward domain II, which itself is rotated toward domain I relative to domain III. Extensive contacts are observed between the amino-terminal domain of EF-Tsmt and domain I of EF-Tumt. Furthermore, the conserved TDFV sequence of EF-Tsmt also contacts domain I with the side chain of Asp139 contacting helix B of EF-Tumt and inserting the side chain of Phe140 between helices B and C. The structure of the EF-Tumt.Tsmt complex provides new insights into the nucleotide exchange mechanism and provides a framework for explaining much of the mutational data obtained for this complex.     

Solution structure of the GUCT domain from human RNA helicase II/Gu beta reveals the RRM fold, but implausible RNA interactions

Human RNA helicase II/Gu alpha (RH-II/Gu alpha) and RNA helicase II/Gu beta (RH-II/Gu beta) are paralogues that share the same domain structure, consisting of the DEAD box helicase domain (DEAD), the helicase conserved C-terminal domain (helicase_C), and the GUCT domain. The N-terminal regions of the RH-II/Gu proteins, including the DEAD domain and the helicase_C domain, unwind double-stranded RNAs. The C-terminal tail of RH-II/Gu alpha, which follows the GUCT domain, folds a single RNA strand, while that of RH-II/Gu beta does not, and the GUCT domain is not essential for either the RNA helicase or foldase activity. Thus, little is known about the GUCT domain. In this study, we have determined the solution structure of the RH-II/Gu beta GUCT domain. Structural calculations using NOE-based distance restraints and residual dipolar coupling-based angular restraints yielded a well-defined structure with beta-alpha-alpha-beta-beta-alpha-beta topology in the region for K585-A659, while the Pfam HMM algorithm defined the GUCT domain as G571-E666. This structure-based domain boundary revealed false positives in the sequence homologue search using the HMM definition. A structural homology search revealed that the GUCT domain has the RRM fold, which is typically found in RNA-interacting proteins. However, it lacks the surface-exposed aromatic residues and basic residues on the beta-sheet that are important for the RNA recognition in the canonical RRM domains. In addition, the overall surface of the GUCT domain is fairly acidic, and thus the GUCT domain is unlikely to interact with RNA molecules. Instead, it may interact with proteins via its hydrophobic surface around the surface-exposed tryptophan.     

Direct interaction of SOS1 Ras exchange protein with the SH3 domain of phospholipase C-gamma1

A recent report that microinjection of the SH3 domain of PLC-gamma1 could induce DNA synthesis raised the functional importance of the SH3 domain of PLC-gamma1 in mitogenic signaling. In this report, we provide evidence that SOS1, a p21Ras-specific guanine nucleotide exchange factor, directly binds to the SH3 domain of PLC-gamma1, and that the SH3 domain of PLC-gamma1 is involved in SOS1-mediated p21Ras activation. SOS1 was coprecipitated with the GST-fused SH3 domain of PLC-gamma1 in vitro. The interaction between SOS1 and the PLC-gamma1 SH3 domain is mediated by direct physical interaction. The carboxyl-terminal proline-rich domain of SOS1 is involved in the interaction with the PLC-gamma1 SH3 domain. Moreover, PLC-gamma1 could be co-immunoprecipitated with SOS1 antibody in cell lysates. From transient expression studies, we could demonstrate that the SH3 domain of PLC-gamma1 is necessary for the association with SOS1 in vivo. Intriguingly, overexpression of the SH3 domain of PLC-gamma1, lipase-inactive PLC-gamma1, or wild-type PLC-gamma1 elevated p21Ras activity and ERK activity when compared with vector transfected cells. The PLC-gamma1 mutant lacking the SH3 domain could not activate p21Ras. p21Ras activities in cell lines overexpressing either PLC-gamma1 or the SH2-SH2-SH3 domain of PLC-gamma1 were elevated about 2-fold compared to vector transfected cells. This study is the first to demonstrate that the PLC-gamma1 SH3 domain enhances p21Ras activity, and that the SH3 domain of PLC-gamma1 may be involved in the SOS1-mediated signaling pathway.     

A new class of signal transducer in His-Asp phosphorelay systems

Nitrate transport activity of the LtnT permease of the cyanobacterium Synechococcus elongatus is activated when LtnA, a response regulator without an effector domain, is phosphorylated by LtnB, a hybrid histidine kinase. We identified a protein (LtnC) that is required for activation of LtnT. LtnC consists of an N-terminal histidine-containing phosphoacceptor (HisKA) domain, a receiver domain, and a unique C-terminal domain found in some cyanobacterial proteins. Because LtnC lacks an ATP-binding kinase domain of a histidine kinase, it is incapable of autophosphorylation, but LtnC is phosphorylated by LtnA. The histidine residue in the HisKA domain but not the aspartate residue in the receiver domain is essential for phosphorylation of LtnC and activation of LtnT. LtnC phosphorylation leads to oligomerization of the protein. Fusion of the C-terminal domain of LtnC to glutathione S-transferase, which forms oligomers, also activates LtnT, suggesting that oligomerization of the LtnC C-terminal domain causes LtnT activation. These results indicate that the C-terminal domain of LtnC acts as an effector domain that directs the output of the signal from the phosphorelay system. The two-step (His-Asp-His) phosphorelay system, composed of the LtnB, LtnA, and LtnC proteins, is distinct from the known phosphorelay systems, namely, the typical two-component system (His-Asp) and the multistep phosphorelay system (His-Asp-His-Asp), because the HisKA domain of LtnC is the terminal phosphoacceptor that determines the signal output. LtnC is a new class of signal transducer in His-Asp phosphorelay systems that contains a HisKA domain and an effector domain.     

孢子体型自交不亲和反应臂重复蛋白ARC1

臂重复蛋白ARC1（arm repeat containing 1,ARC1）是孢子体型自交不亲和信号传导途径下游非常重要的蛋白质因子,由臂重复结构域、U-box结构域、亮氨酸拉链,卷曲螺旋结构域、1个核定位信号和2个核输出信号组成,其中臂重复结构域和U—box结构域起主要功能。ARC1具有E3泛素连接酶活性,能够促进自交不亲和反应（self-incompatibility,SI）中的信号传导元件泛素化并降解。本文综述了ARC1蛋白的结构和功能,旨在阐明它在SI反应中的作用。     

Posttranslational processing and differential glycosylation of Tractin, an Ig-superfamily member involved in regulation of axonal outgrowth

Tractin is a novel member of the Ig-superfamily which has a highly unusual structure. It contains six Ig domains, four FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine-rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ domain binding motif. By generating domain-specific antibodies, we show that Tractin is proteolytically processed at two cleavage sites, one located in the third FNIII domain, and a second located just proximal to the transmembrane domain resulting in the formation of four fragments. The most NH(2)-terminal fragment which is glycosylated with the Lan3-2, Lan4-2, and Laz2-369 glycoepitopes is secreted, and we present evidence which supports a model in which the remaining fragments combine to form a secreted homodimer as well as a transmembrane heterodimer. The extracellular domain of the dimers is mostly made up of the collagen-like PG/YG-repeat domain but also contains 11/2 FNIII domain and the acidic domain. The collagen-like PG/YG-repeat domain could be selectively digested by collagenase and we show by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin. Thus, the transmembrane heterodimer of Tractin constitutes a novel protein domain configuration where sequence that has properties similar to that of extracellular matrix molecules is directly linked to the cytoskeleton through interactions with ankyrin.     

The YARHG domain: an extracellular domain in search of a function

We have identified a new bacterial protein domain that we hypothesise binds to peptidoglycan. This domain is called the YARHG domain after the most highly conserved sequence-segment. The domain is found in the extracellular space and is likely to be composed of four alpha-helices. The domain is found associated with protein kinase domains, suggesting it is associated with signalling in some bacteria. The domain is also found associated with three different families of peptidases. The large number of different domains that are found associated with YARHG suggests that it is a useful functional module that nature has recombined multiple times.     

Solution and membrane-bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain

During cell intoxication by diphtheria toxin, endosome acidification triggers the translocation of the catalytic (C) domain into the cytoplasm. This event is mediated by the translocation (T) domain of the toxin. Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin. Using partitioning experiments with lipid vesicles, fluorescence spectroscopy, and a lipid vesicle leakage assay, we characterized the dominant behavior of the T domain over the C domain during the successive steps by which these domains interact with a membrane upon acidification: partial unfolding in solution and during membrane binding, and then structural rearrangement during penetration into the membrane. To this end, we compared, for each domain, isolated or linked together in a CT protein (the toxin lacking the receptor-binding domain), each of these steps. The behavior of the T domain is marginally modified by the presence or absence of the C domain, whereas that of the C domain is greatly affected by the presence of the T domain . All of the steps leading to membrane penetration of the C domain are triggered at higher pH by the T domain , by 0.5-1.6 pH units. The T domain stabilizes the partially folded states of the C domain corresponding to each step of the process. The results unambiguously demonstrate that the T domain acts as a specialized pH-dependent chaperone for the C domain. Interestingly, this chaperone activity acts on very different states of the protein: in solution, membrane-bound, and membrane-inserted.     

Conformation and domain structure of the non-histone chromosomal proteins HMG 1 and 2. Domain interactions

The sequence of the 224 residues of HMG 1 suggests it consists of three domains. We have previously proposed [Cary et al. (1980 Eur. J. Biochem. 131, 367-374] that the A and B domains can fold autonomously and that there is also a small N domain. Several proteases are now found to cut at the end of the B domain (at or close to residue 184). It is shown that the A + B-domain fragment also folds and probably contains all the helix of intact HMG 1. The stability of the B domain is enhanced by the presence of the A domain. The acidic C domain undergoes a coil----helix transition on lowering the pH. Several peptides have been prepared by cleavage at tryptophan. Peptide 57--C-terminus contains complete B and C domains but does not fold. In the absence of the A domain the C domain is thus able to destabilise the B domain. It is concluded that the stability of the B domain in HMG 1 is due to interaction with the A domain and the C domain has a separate function from the other domains.