Intramolecular Interactions between the Protease and Structural Domains Are Important for the Functions of Serine Protease Autotransporters

ABSTRACT Autotransporter (AT) is a protein secretion pathway found in Gram-negative bacteria featuring a multidomain polypeptide with a signal sequence, a passenger domain, and a translocator domain. An AT subfamily named serine protease ATs of the family Enterobacteriaceae (SPATEs) is characterized by the presence of a conserved serine protease motif in the passenger domain which contributes to bacterial pathogenesis. The goal of the current study is to determine the importance of the passenger domain conserved residues in the SPATE proteolytic and adhesive functions using the temperature-sensitive hemagglutinin (Tsh) protein as our model. To begin, mutations of 21 fully conserved residues in the four passenger domain conserved motifs were constructed by PCR-based site-directed mutagenesis. Seventeen mutants exhibited a wild-type secretion level; among these mutants, eight displayed reduced proteolytic activities in Tsh-specific oligopeptide and mucin cleavage assays. These eight mutants also demonstrated lower affinities to extracellular matrix proteins, collagen IV, and fibronectin. These eight conserved residues were analyzed by molecular graphics modeling to demonstrate their intramolecular interactions with the catalytic triad and other key residues. Additional mutations were made to confirm the above interactions in order to demonstrate their significance to the SPATE functions. Altogether our data suggest that certain conserved residues in the SPATE passenger domain are important for both the proteolytic and adhesive activities of SPATE by maintaining the proper protein structure via intramolecular interactions between the protease and β-helical domains. Here, we provide new insight into the structure-function relationship of the SPATEs and the functional roles of their conserved residues.

Download Full-text

Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

Turkish Journal of Biochemistry ◽

10.1515/tjb-2016-0169 ◽

2016 ◽

Vol 42 (2) ◽

Author(s):

Hasnain Hussain ◽

Nikson Fatt Ming Chong

Keyword(s):

Catalytic Activity ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Qualitative And Quantitative Analysis ◽

Conserved Residues ◽

Overlap Extension Pcr ◽

Overlap Extension ◽

Catalytic Network ◽

And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

Download Full-text

Importance of Conserved Residues of the Serine Protease Autotransporter β-Domain in Passenger Domain Processing and β-Barrel Assembly

Infection and Immunity ◽

10.1128/iai.00390-10 ◽

2010 ◽

Vol 78 (8) ◽

pp. 3516-3528 ◽

Cited By ~ 12

Author(s):

Yihfen T. Yen ◽

Casey Tsang ◽

Todd A. Cameron ◽

Dennis O. Ankrah ◽

Athina Rodou ◽

...

Keyword(s):

Outer Membrane ◽

Serine Protease ◽

Cell Fractionation ◽

Passenger Domain ◽

Double Mutation ◽

Conserved Residues ◽

Virulence Proteins ◽

Secretion Pathway ◽

Residue Substitution ◽

Cellular Compartments

ABSTRACT Serine protease autotransporters of the family Enterobacteriaceae (SPATE) comprise a family of virulence proteins secreted by enteric Gram-negative bacteria via the autotransporter secretion pathway. A SPATE polypeptide contains a C-terminal translocator domain that inserts into the bacterial outer membrane as a β-barrel structure and mediates secretion of the passenger domain to the extracellular environment. In the present study, we examined the role of conserved residues located in the SPATE β-barrel-forming region in passenger domain secretion. Thirty-nine fully conserved residues in Tsh were mutated by single-residue substitution, and defects in their secretion phenotypes were assessed by cell fractionation and immunochemistry. A total of 22 single mutants exhibited abnormal phenotypes in different cellular compartments. Most mutants affecting secretion are charged residues with side chains pointing into the β-barrel interior. Seven mutants showed notable abnormalities in processing (constructs with the E1231A, E1249A, and R1374A mutations) and β-barrel assembly or insertion into the outer membrane (constructs with the G1158Y, F1360A, Y1375A, and F1377A mutations). The phenotypes of the β-barrel assembly/insertion mutants and the presence of a processed Tsh passenger domain in the periplasm support the possibility that the translocator domain must undergo extensive folding prior to insertion into the outer membrane. Results from double-mutation experiments further demonstrate that F1360 and F1377 affect β-barrel insertion/assembly at different times. In light of these new data, a more refined model for the mechanism of SPATE secretion is presented.

Download Full-text

Functional Analysis of the Tsh Autotransporter from an Avian Pathogenic Escherichia coli Strain

Infection and Immunity ◽

10.1128/iai.72.10.5548-5554.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5548-5554 ◽

Cited By ~ 48

Author(s):

Maria Kostakioti ◽

Christos Stathopoulos

Keyword(s):

Escherichia Coli ◽

Serine Protease ◽

Collagen Iv ◽

Binding Activity ◽

Coli Strain ◽

Matrix Proteins ◽

Temperature Sensitive ◽

Avian Pathogenic Escherichia Coli ◽

Passenger Domain ◽

Pathogenic Escherichia Coli

ABSTRACT The temperature-sensitive hemagglutinin (Tsh) is an autotransporter protein secreted by avian-pathogenic Escherichia coli strains that colonize the respiratory tract and lead to airsacculitis, pericarditis, and colisepticemia. It is synthesized as a 140-kDa precursor protein, whose processing results in a 106-kDa passenger domain (Tshs) and a 33-kDa β-domain (Tshβ). The presence of a conserved 7-amino-acid serine protease motif within Tshs classifies the protein in a subfamily of autotransporters, known as serine protease autotransporters of the Enterobacteriaceae. In this study, we report that purified Tshs is capable of adhering to red blood cells, hemoglobin, and the extracellular matrix proteins fibronectin and collagen IV. We also demonstrate that Tshs exerts proteolytic activity against casein, and we provide experimental evidence demonstrating that serine 259 is essential for the protease function. However, this residue is not required for adherence to substrates, and its replacement by an alanine does not abolish binding activity. In summary, our results demonstrate that Tsh is a bifunctional protein with both adhesive and proteolytic properties.

Download Full-text

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.03666-12 ◽

2013 ◽

Vol 79 (9) ◽

pp. 2968-2978 ◽

Cited By ~ 14

Author(s):

Aamir Ghafoor ◽

Zoe Jordens ◽

Bernd H. A. Rehm

Keyword(s):

Pseudomonas Aeruginosa ◽

Glucose Dehydrogenase ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Conserved Residues ◽

Content Type ◽

Functional Roles ◽

Polysaccharide Biosynthesis

ABSTRACTPseudomonas aeruginosaproduces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes,pelAtopelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenicpelFdeletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally,in vitroexperiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX7E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ± 3.15% (mean ± standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.

Download Full-text

Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of Enterobacteriaceae

Journal of Bacteriology ◽

10.1128/jb.00754-09 ◽

2009 ◽

Vol 191 (21) ◽

pp. 6571-6583 ◽

Cited By ~ 88

Author(s):

Fernando Ruiz-Perez ◽

Ian R. Henderson ◽

Denisse L. Leyton ◽

Amanda E. Rossiter ◽

Yinghua Zhang ◽

...

Keyword(s):

Serine Protease ◽

Protein Interactions ◽

Hybrid Approach ◽

Large Family ◽

Site Directed Mutagenesis ◽

Passenger Domain ◽

Surface Plasmon Resonance Analysis ◽

C Terminus ◽

Mutant Strains ◽

Periplasmic Chaperones

ABSTRACT The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a β-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by Escherichia coli O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-β or EspP passenger domains. The secretion of EspP was moderately reduced in the surA and skp mutant strains but severely impaired in the degP background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in ∼80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct protein-protein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker “autotransporter.”

Download Full-text

Direct evidence of a low barrier hydrogen bond in the catalytic triad of a Serine protease

Scientific Reports ◽

10.1038/s41598-018-28441-7 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 11

Author(s):

Peter Agback ◽

Tatiana Agback

Keyword(s):

Hydrogen Bond ◽

Serine Protease ◽

Direct Evidence ◽

Catalytic Triad

Download Full-text

SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2154 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2154-2164 ◽

Cited By ~ 50

Author(s):

D J DeMarini ◽

M Winey ◽

D Ursic ◽

F Webb ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Gene Product ◽

Signal Sequence ◽

Null Alleles ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

Download Full-text

Functional Roles of Conserved Residues in the Unstructured Loop ofVibrio harveyiBacterial Luciferase†

Biochemistry ◽

10.1021/bi011958p ◽

2002 ◽

Vol 41 (6) ◽

pp. 1724-1731 ◽

Cited By ~ 12

Author(s):

John C. Low ◽

Shiao-Chun Tu

Keyword(s):

Conserved Residues ◽

Functional Roles

Download Full-text

Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3872-3881.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3872-3881

Author(s):

F Estruch ◽

M Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Wilms Tumor ◽

Temperature Sensitive ◽

Carbon Utilization ◽

Functional Roles ◽

Multicopy Suppressor ◽

Finger Protein

The MSN2 gene was selected as a multicopy suppressor in a temperature-sensitive SNF1 protein kinase mutant of Saccharomyces cerevisiae. MSN2 encodes a Cys2His2 zinc finger protein related to the yeast MIG1 repressor and to mammalian early growth response and Wilms' tumor zinc finger proteins. Deletion of MSN2 caused no phenotype. A second similar zinc finger gene, MSN4, was isolated, and deletion of both genes caused phenotypic defects related to carbon utilization. Overexpression of the zinc finger regions was deleterious to growth. LexA-MSN2 and LexA-MSN4 fusion proteins functioned as strong transcriptional activators when bound to DNA. Functional roles of this zinc finger protein family are discussed.

Download Full-text