Protein secretion is a major contributor to Gram-negative bacterial virulence. Type Vb or two-partner secretion (TPS) pathways utilize a membrane bound b-barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C-proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N-proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site-specific mutants of HpmA265. By correlating the effect of individual site-specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C-terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the Abbreviations: CD, circular dichroism; C m , guanidine hydrochloride concentration at transition mid-point; FHA, Bordetella pertussis filamentous hemagglutinin; D, denatured HpmA265 TPS domain; GdnHCl, guanidine•HCl; HpmA, Proteus mirabilis full length hemolysin A; HpmA265, truncation fragment of P. mirabilis hemolysin A processed to start at asparagine 30 and cloned to end at glycine 265; I 1 and I 2 , HpmA265 unfolding intermediates 1 and 2; MALDI-TOF MS, matrix-assisted laser desorption/ ionization time of flight mass spectrometry; N, native HpmA265 TPS domain; PBS, phosphate buffered saline; POTRA domain, polypeptide-transport associated domain; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC-LS, sizeexclusion chromatography light scattering; SD1, SD2, SD3, structural subdomains within the HpmA265 TPS domain; T 1 , T 2 , T 3 , transitions 1, 2, and 3 within the HpmA265 TPS domain four-state unfolding model; TPS, two-partner secretion; TpsA, two-partner secretion pathway A component; TpsB, two-partner secretion pathway B component; Vmax, maximum velocity.Additional Supporting Information may be found in the online version of this article.Megan R. Wimmer and Christopher N. Woods contributed equally to this work. folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C-terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N-proximal polar core subdomain.
Wild-type and variant forms of HpmA265 (truncated hemolysin A) from Proteus mirabilis reveal a right-handed, parallel -helix capped and flanked by segments of antiparallel -strands. The low-salt crystal structures form a dimeric structure via the implementation of on-edge main-chain hydrogen bonds donated by residues 243-263 of adjacent monomers. Surprisingly, in the high-salt structures of two variants, Y134A and Q125A-Y134A, a new dimeric interface is formed via main-chain hydrogen bonds donated by residues 203-215 of adjacent monomers, and a previously unobserved tetramer is formed. In addition, an eight-stranded antiparallel -sheet is formed from the flap regions of crystallographically related monomers in the high-salt structures. This new interface is possible owing to additional proteolysis of these variants after Tyr240. The interface formed in the high-salt crystal forms of hemolysin A variants may mimic the on-edge -strand positioning used in template-assisted hemolytic activity.
Small organic molecules, like phenylalanine and theophylline, are effective inhibitors of mammalian alkaline phosphatases, such as calf intestinal alkaline phosphatase (CIAP). However, organic compounds do not hamper E. coli alkaline phosphatase (EcAP) activity. Sequence and structural analysis of alkaline phosphatase isozymes revealed a lack of conservation at EcAP residues that may be important for organic inhibition, thereby providing a potential explanation for the contrary inhibition. By mutating these EcAP residues to mimic analogous residues in mammalian APases, uncompetitive inhibition of EcAP by a class of aromatic molecules was conferred. While variants with single mutations are unaffected by organic effectors, variants expressing multiple mutations are inhibited, suggesting a synergistic relationship essential for organic binding. Circular dichroism was utilized to verify similar stability and kinetics for each variant. Michaelis‐Menten experiments were used to identify inhibition and confirm similar pH and cofactor dependence. The importance of key residues was determined based on their ability to confer organic inhibition. Furthermore, utilization of a set of purine derivatives allowed determination of the structure‐activity relationship for inhibitor binding. Specifically, this analysis enabled identification of hydrogen bonding requirements for organic inhibition. In total, designed substitutions altering EcAP residues near the active site to mimic analogous residues in mammalian APases confers uncompetitive inhibition by specific derivatives of a class of aromatic organic molecules.Support or Funding InformationResearch generously supported by a University of Wisconsin ‐ La Crosse Undergraduate Research and Creativity grant (MRM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
As bacterial pathogens gain antibacterial resistance, few mechanisms remain available for halting pathogenicity, giving a frightening glimpse into a post‐antibacterial era. However, the secretion of virulence factors is often imperative to bacterial pathogenesis within the human body and is becoming a larger target for antibiotic treatment. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system currently known. In fact, whooping cough, meningitis, UTIs, and certain food‐borne illnesses have been attributed to gram‐negative bacterial species containing the TPS system. Systematically, TpsA members are activated concomitant with TpsB‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration thus allowing the pathogen to invade and proliferate within the host, advantageously. Structurally, TpsA members can be divided into a TPS domain and a functional (virulent) domain. All TPS domains are constructed from a ~300‐residue right‐handed, parallel, β‐helix, and recognize their cognate TpsB membrane‐bound partner during secretion. Fundamentally, TpsA β‐helix structures are built from consecutive β‐circuits, where each β‐circuit is constructed from three parallel β‐strands. In order to further understand the relationship between complete TpsA β‐circuit establishment, structural stability and function we have implemented a truncated form of hemolysin A (HpmA265) from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Structurally, the carboxy‐terminal subdomain harbors a partial, two‐stranded β‐circuit. Previous investigations replaced valine 158 (V158) and phenylalanine 215 (F215), as located within the first and last parallel β‐strands of the non‐polar core subdomain, with polar residues. The site‐selective alteration of V158 demonstrated increased function, while merging the unfolding transitions associated with the non‐polar and carboxy‐terminal subdomains. Alternatively, site‐selective alteration of F215 demonstrated decreased function, while destabilizing the transitions associated with both the non‐polar and carboxy‐terminal subdomains. Ultimately, template‐assisted activity has been interrelated to extent of β‐circuit structural destabilization within the carboxy‐terminal subdomain. Specifically, this research expands upon the previous V158 and F215 results by probing the structural and functional relationship via progressive amino‐acid extension to the HpmA265 carboxy‐terminal subdomain. Ultimately, the structural and functional effects of final β‐circuit completion will be ascertained using protein unfolding and hemolytic functional measurements.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The secretion of virulence factors often aids bacterial pathogenesis. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system. In fact, whooping cough, meningitis, and certain food‐borne illnesses have been attributed to TPS pathway containing gram‐negative bacterial species. Systematically, TpsA members are activated concomitant with Tps‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration. Collectively, these TpsA functions provide advantageous invasion and proliferation strategies within host cells. Structurally, TpsA members can be divided into a two‐partner secretion (TPS) domain and a functional domain. All TPS domains are constructed from a 300‐residue right‐handed, parallel β‐helix structure, and recognize their cognate TpsB partner. In order to further understand the relationship between TPS domains and TpsA structure and function, we have implemented a truncated form of hemolysin A (HpmA265), a TpsA member from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Specifically, these research investigations targeted valine 158 (V158) and phenylalanine 215 (F215) located within the first and last parallel β‐strands of the non‐polar core subdomain. A series of site‐selective variations were established at both V158 and F215. These variant forms of HpmA265 were characterized structurally via protease sensitivity and protein folding techniques, while functionality was ascertained within a template‐assisted hemolysis assay. Structurally, the V158 variants have destabilized the unfolding transitions associated with both the polar and non‐polar core subdomains, while leaving functionality unaffected. Site‐selective variants at F215 have selectively destabilized the non‐polar core subdomain, while leaving the unfolding transition attributed to the polar core subdomain unaffected. Additionally, the F215 variants do not affect template‐assisted hemolysis. Therefore, our results have been able to dissect the structural stability within the non‐polar core subdomain from template‐assisted function. These results have expanded the understanding for the implementation of TPS domains within gram‐negative bacteria.Support or Funding InformationFunding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (JDG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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