The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB

The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two-hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C-terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was confirmed by cross-linking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans-envelope complex that brings the inner and outer membranes in close proximity.     

The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA)

As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.     

Effect of the C-terminal domains and terminal residues of catalytic domain on enzymatic activity and thermostability of lichenase from Clostridium thermocellum

To elucidate the effects of C-terminal domains of LicMB (mature lichenase from Clostridium thermocellum) and terminal residues of LicMB-CD (catalytic domain of LicMB) on the properties of lichenase, a series of truncated genes were constructed and expressed in E. coli. The Thr-Pro box had a positive effect while the dockerin domain had a negative impact on the properties of LicMB. The N-terminal 10–25th and C-terminal 1–9th residues of LicMB-CD were necessary to retain high thermostability while the N-terminal 1–7th and C-terminal 1–3rd residues were not necessary to maintain enzymatic activity.     

Identification of three key active site residues in the C-terminal domain of human recombinant folylpoly-gamma-glutamate synthetase by site-directed mutagenesis.

Three cysteines in human recombinant folylpoly-gamma-glutamate synthetase (FPGS) that were reactive with iodoacetamide were located in peptides that were highly conserved across species; the functions of two of these peptides, located in the C-terminal domain, were studied by site-directed mutagenesis. When cDNAs containing mutations in each conserved ionic residue on these peptides were transfected into AUXB1 cells, which lack endogenous FPGS activity, one mutant (D335A) did not complement the auxotrophy, and another (R377A) allowed only minimal growth. FPGS activity could not be detected in insect cells expressing abundant levels of these two mutant proteins from recombinant baculoviruses nor from a virus encoding an H338A mutant FPGS. Kinetic analysis of the purified proteins demonstrated that each of these three mutants was quite different from the others. The major kinetic change detected for the H338A mutation was a 600-fold increase in the K(m) for glutamic acid. For the D335A mutation, the binding of all three substrates (aminopterin, ATP, and glutamic acid) was affected. For R377A, the K(m) for glutamic acid was increased by 1500-fold, and there was an approximately 20-fold decrease in the k(cat) of the reaction. The binding of the K(+) ion, a known activator of FPGS, was affected by the D335A and H338A mutations. We conclude that these three amino acids participate in the alignment of glutamic acid in the active site and that Arg-377 is also involved in the mechanism of the reaction.     

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.     

Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.     

Structural requirements for key residues and auxiliary portions of a BLUF domain

BlrB in Rhodobacter sphaeroides is a single domain, flavin-based blue light sensor protein in the BLUF family of photoreceptors. Consistent with other members of this family, blue light excitation induces a putative signaling state characterized by a 10 nm red shift in the UV-visible absorbance spectrum. Structural and spectroscopic characterization of truncated BlrB constructs establishes that the C-terminal 50 amino acids of this protein are essential to its structural integrity despite not being part of the canonical BLUF domain architecture. Mutagenesis studies support the critical roles of Tyr9, Asn33, and Gln51 for flavin binding and the integrity of the BLUF domain fold. Comparison of solution NMR spectra of BlrB acquired under dark and light conditions indicates very limited light-dependent conformational changes except for a few interesting residues: Trp92, Met94, and Ile127. Notably, the Ile127 side chain experiences significant chemical shift changes despite the fact that it is far ( approximately 15 A) from the flavin chromophore in the C-terminal extension. These data suggest that the light-induced signal is propagated from the flavin through the beta sheet to the last two alpha helices in the C-terminal extension, potentially providing a mechanism to transmit this change to initiate a cellular response to blue light.     

Evidence for the participation of histidine residues located in the 56 kDa C-terminal polypeptide domain of ADP-ribosyl transferase in its catalytic activity

Purified ADPRT protein was inactivated by the histidine specific reagent diethylpyrocarbonate, binding to two histidine residues, or by a relatively histidine selective photoinactivation method. Inactivation with up to 1.3 mM diethylpyrocarbonate was reversible by hydroxylamine. Enzymatic inactivation coincided with the loss of binding capacity of the enzyme protein to benzamide affinity matrix but not to DNA cellulose. Labelled diethylpyrocarbonate was identified exclusively in the 56 kDa carboxyl-terminal polypeptide where 2 out of 13 histidine residues were modified by this reagent. It is proposed that histidine residues in the 56 kDa polypeptide may participate as initiator sites for polyADP-ribosylation.     

C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.     

Phosphorylation of a C-terminal auto-inhibitory domain increases SMARCAL1 activity

SMARCAL1 promotes the repair and restart of damaged replication forks. Either overexpression or silencing SMARCAL1 causes the accumulation of replication-associated DNA damage. SMARCAL1 is heavily phosphorylated. Here we identify multiple phosphorylation sites, including S889, which is phosphorylated even in undamaged cells. S889 is highly conserved through evolution and it regulates SMARCAL1 activity. Specifically, S889 phosphorylation increases the DNA-stimulated ATPase activity of SMARCAL1 and increases its ability to catalyze replication fork regression. A phosphomimetic S889 mutant is also hyperactive when expressed in cells, while a non-phosphorylatable mutant is less active. S889 lies within a C-terminal region of the SMARCAL1 protein. Deletion of the C-terminal region also creates a hyperactive SMARCAL1 protein suggesting that S889 phosphorylation relieves an auto-inhibitory function of this SMARCAL1 domain. Thus, S889 phosphorylation is one mechanism by which SMARCAL1 activity is regulated to ensure the proper level of fork remodeling needed to maintain genome integrity during DNA synthesis.     

Regulation of insulin-regulated membrane aminopeptidase activity by its C-terminal domain

The development of inhibitors of insulin-regulated aminopeptidase (IRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that associated with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homology models based on the structures of recently determined related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examined their kinetic and biochemical properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments containing domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that IRAP has a second Zn(2+) binding site, not associated with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP.     

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.     

Basic residues in the Mason-Pfizer monkey virus gag matrix domain regulate intracellular trafficking and capsid-membrane interactions

Mason-Pfizer monkey virus (M-PMV) capsids that have assembled in the cytoplasm must be transported to and associate with the plasma membrane prior to being enveloped by a lipid bilayer during viral release. Structural studies have identified a positive-charge density on the membrane-proximal surface of the matrix (MA) protein component of the Gag polyprotein. To investigate if basic amino acids in MA play a role in intracellular transport and capsid-membrane interactions, mutants were constructed in which lysine and arginine residues (R10, K16, K20, R22, K25, K27, K33, and K39) potentially exposed on the capsid surface were replaced singly and in pairs by alanine. A majority of the charge substitution mutants were released less efficiently than the wild type. Electron microscopy of mutant Gag-expressing cells revealed four distinct phenotypes: K16A and K20A immature capsids accumulated on and budded into intracellular vesicles; R10A, K27A, and R22A capsid transport was arrested at the cellular cortical actin network, while K25A immature capsids were dispersed throughout the cytoplasm and appeared to be defective at an earlier stage of intracellular transport; and the remaining mutant (K33A and K39A) capsids accumulated at the inner surface of the plasma membrane. All mutants that released virions exhibited near-wild-type infectivity in a single-round assay. Thus, basic amino acids in the M-PMV MA define both cellular location and efficiency of virus release.     

Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.

The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.     

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues; highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T(191) and Y(193) in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T(187) showed reduced enzymatic activity against exogenous substrates but retained autophosphorylation activity. Phosphorylation was confirmed by the use of a phospho-specific T(187) antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed.     

Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex

The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex.