scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

On mechanisms of colicin import: the outer membrane quandary

2018 ◽  
Vol 475 (23) ◽  
pp. 3903-3915 ◽  
Author(s):  
William A. Cramer ◽  
Onkar Sharma ◽  
S.D. Zakharov
Keyword(s):  
Outer Membrane ◽  
Catalytic Domain ◽  
Efflux Pump ◽  
Crystal Structure Analysis ◽  
Multidrug Efflux Pump ◽  
Terminal Domain ◽  
Colicin E1 ◽  
N Terminus ◽  
T Domain

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10−9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

scholarly journals Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

2011 ◽  
Vol 22 (2) ◽  
pp. 189-201 ◽  
Author(s):  
Roman Gorelik ◽  
Changsong Yang ◽  
Vasumathi Kameswaran ◽  
Roberto Dominguez ◽  
Tatyana Svitkina
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Coincidence Detection ◽  
Membrane Targeting ◽  
Amino Terminal ◽  
N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

scholarly journals NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

2008 ◽  
Vol 105 (40) ◽  
pp. 15287-15292 ◽  
Author(s):  
Rodney E. Infante ◽  
Michael L. Wang ◽  
Arun Radhakrishnan ◽  
Hyock Joo Kwon ◽  
Michael S. Brown ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lipid Bilayers ◽  
Clinical Phenotype ◽  
Phosphatidylcholine Liposomes ◽  
Terminal Domain ◽  
Naturally Occurring ◽  
Membrane Bound ◽  
Niemann Pick ◽  
Lysosomal Proteins

Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann–Pick C1 (NPC1) and soluble Niemann–Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25–264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [3H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.

scholarly journals Set2-Dependent K36 Methylation Is Regulated by Novel Intratail Interactions within H3

2009 ◽  
Vol 29 (24) ◽  
pp. 6413-6426 ◽  
Author(s):  
James N. Psathas ◽  
Suting Zheng ◽  
Song Tan ◽  
Joseph C. Reese
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Transcription Initiation ◽  
Gene Activation ◽  
N Terminus

ABSTRACT Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.

scholarly journals Intra-helical salt bridge contribution to membrane protein insertion

2021 ◽  
Author(s):  
Gerard Duart ◽  
John Lamb ◽  
Arne Elofsson ◽  
Ismael Mingarro
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Electrostatic Interactions ◽  
Salt Bridge ◽  
Protein Stabilization ◽  
Membrane Insertion ◽  
Salt Bridges ◽  
Protein Insertion

ABSTRACTSalt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a very hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. This can sometimes be avoided by TM region rearrangements after insertion, but it is also possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we use an analytical pipeline to study the prevalence of charged pairs of amino acid residues in TM α-helices, which shows that potentially salt-bridge forming pairs are statistically over-represented. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and in vivo systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute between 0.5-0.7 kcal/mol to the apparent free energy of membrane insertion (ΔGapp). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.

scholarly journals Chloride channel activity of ClC-2 is modified by the actin cytoskeleton

2000 ◽  
Vol 352 (3) ◽  
pp. 789-794 ◽  
Author(s):  
Najma AHMED ◽  
Mohabir RAMJEESINGH ◽  
Simeon WONG ◽  
Alison VARGA ◽  
Elizabeth GARAMI ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Chloride Channel ◽  
Electrostatic Interactions ◽  
Osmotic Shock ◽  
Expression System ◽  
Cell Volume Regulation ◽  
Channel Activity ◽  
N Terminus ◽  
Inhibitory Domain

The chloride channel ClC-2has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel.

scholarly journals Identification of Amino Acids That Are Critical to the Processivity Function of Respiratory Syncytial Virus M2-1 Protein

2003 ◽  
Vol 77 (9) ◽  
pp. 5046-5053 ◽  
Author(s):  
Helen Zhou ◽  
Xing Cheng ◽  
Hong Jin
Keyword(s):  
Amino Acids ◽  
Respiratory Syncytial Virus ◽  
Amino Acid Identity ◽  
Chimeric Proteins ◽  
Acid Identity ◽  
Level Of Activity ◽  
N Terminus ◽  
Processivity Factor ◽  
Syncytial Virus

ABSTRACT The M2-1 protein of respiratory syncytial virus (RSV) is a transcription processivity factor that is essential for virus replication. The function of RSV M2-1 protein can be examined by using an RSVlacZ minigenome assay in vitro since the expression of the lacZ gene is dependent on M2-1. The M2-1 protein of pneumonia virus of mice (PVM), also a member of the Pneumovirus genus, functions poorly in the RSVlacZ minigenome assay despite conservation of the Cys3-His1 motif at its N terminus and an overall 40% amino acid identity with RSV M2-1. To identify the amino acids responsible for the differences between these two proteins, two chimeric proteins were constructed. The RSV/PVM (RP) M2-1 chimera that contains the N-terminal 30 amino acids from RSV and the remaining C-terminal 148 amino acids from PVM maintained a level of activity at an ca. 36% of RSV M2-1. However, the PVM/RSV (PR) M2-1 chimera with the N-terminal 29 amino acids from PVM and 164 amino acids from RSV had an activity of <5% of RSV M2-1, indicating that the functional determinants are mainly located in the N terminus of M2-1. Mutagenesis of the N terminus of PR M2-1 and RSV M2-1 identified that Leu-16 and Asn-17 of RSV M2-1 are critical to the M2-1 function. In addition, several charged residues in the N terminus of RSV M2-1 also contributed to the functional integrity of M2-1.

NUP98-Topoisomerase I acute myeloid leukemia-associated fusion gene has potent leukemogenic activities independent of an engineered catalytic site mutation

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1127-1136 ◽  
Author(s):  
Rhonna M. Gurevich ◽  
Peter D. Aplan ◽  
R. Keith Humphries
Keyword(s):  
Bone Marrow ◽  
Topoisomerase I ◽  
Fusion Gene ◽  
Chromosomal Rearrangements ◽  
Fusion Partner ◽  
Competitive Growth ◽  
Site Mutation ◽  
Murine Bone Marrow ◽  
Cell Counts

AbstractChromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies, resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98, and more than one half of these are homeobox transcription factors. By contrast, the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1), a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors, evidenced by a competitive growth advantage in liquid culture, increased replating efficient of colony-forming cells (CFCs), and a marked increase in spleen colony-forming cell output. Moreover, in a murine bone marrow transplantation model, NUP98-TOP1 expression led to a lethal, transplantable leukemia characterized by extremely high white cell counts, splenomegaly, and mild anemia. Strikingly, a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings, together with similar biologic effects reported for NUP98-HOX fusions, suggest unexpected, overlapping functions of NUP98 fusion genes, perhaps related to common DNA binding properties. (Blood. 2004;104:1127-1136)

scholarly journals Characterization of a New Bacteriocin, Carocin D, from Pectobacterium carotovorum subsp. carotovorum Pcc21

2010 ◽  
Vol 76 (22) ◽  
pp. 7541-7549 ◽  
Author(s):  
Eunjung Roh ◽  
Tae-Ho Park ◽  
Myung-il Kim ◽  
Seungdon Lee ◽  
Sangryeol Ryu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Antibacterial Activity ◽  
Biological Control Agent ◽  
Soft Rot ◽  
Control Agent ◽  
Indicator Strain ◽  
Amino Acid Sequences ◽  
Pectobacterium Carotovorum ◽  
N Terminus

ABSTRACT Two different bacteriocins, carotovoricin and carocin S1, had been found in Pectobacterium carotovorum subsp. carotovorum, which causes soft-rot disease in diverse plants. Previously, we reported that the particular strain Pcc21, producing only one high-molecular-weight bacteriocin, carried a new antibacterial activity against the indicator strain Pcc3. Here, we report that this new antibacterial activity is due to a new bacteriocin produced by strain Pcc21 and named carocin D. Carocin D is encoded by the caroDK gene located in the genomic DNA together with the caroDI gene, which seems to encode an immunity protein. N-terminal amino acid sequences of purified carocin D were determined by Edman degradation. In comparison with the primary translation product of caroDK, it was found that 8 amino acids are missing at the N terminus. This finding proved that carocin D is synthesized as a precursor peptide and that 8 amino acids are removed from its N terminus during maturation. Carocin D has two putative translocation domains; the N-terminal and C-terminal domains are homologous to those of Escherichia coli colicin E3 and Pseudomonas aeruginosa S-type pyocin, respectively. When caroDK and caroDI genes were transformed into carocin D-sensitive bacteria such as Pcc3, the bacteria became resistant to this bacteriocin. Carocin D has one putative DNase domain at the extreme C terminus and showed DNase activity in vitro. This bacteriocin had slight tolerance to heat but not to proteases. The caroDK gene was present in only 5 of 54 strains of P. carotovorum subsp. carotovorum. These results indicate that carocin D is a third bacteriocin found in P. carotovorum subsp. carotovorum, and this bacteriocin can be readily expressed in carocin D-sensitive nonpathogenic bacteria, which may have high potential as a biological control agent in the field.

scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

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