Abstract
The disulfide bond signal sequence (DsbAss) protein is characterized as an important virulence factor in gram-negative bacteria. This study aimed to analyze the “alanine” alteration in the hydrophobic (H) region of DsbAss and to understand the conformational DsbAss alteration(s) inside the fifty-four homolog (Ffh)-binding groove which were revealed to be crucial for translocation of ovine growth hormone (OGH) to the periplasmic space in Escherichia coli via the secretory (Sec) pathway. An experimental design was used to explore the hydrophobicity and alteration of alanine (Ala) to isoleucine (Ile) in the tripartite structure of DsbAss. As a result, two DsbAss mutants (Ala at positions -11 and -13) with same hydrophobicity of 1.539 led to the conflicting translocation of the active OGH gene. We performed molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations to examine the interaction energetic and dynamic aspects of DsbAss/signal repetition particle 54 (SRP54) binding, which has a principle role in Escherichia coli Sec pathways. Although both DsbAss mutants retained helicity, the MD simulation analysis evidenced that altering Ala-13 changed the orientation of the signal peptide in the Ffh M binding domain groove, favored more stable interaction energies (MM-GBSA ΔGtotal = −140.62 kcal mol−1), and hampered the process of OGH translocation, while Ala-11 pointed outward due to unstable conformation and less binding energy (ΔGtotal = −124.24 kcal mol−1). Here we report the dynamic behavior of change of “alanine” in the H-domain of DsbAss which affects the process of translocation of OGH, where MD simulation and MM-GBSA can be useful initial tools to investigate the virulence of bacteria.
Highlights
Almost all previously treatable infections have developed antibiotic resistance, leading to “multidrug-resistant pathogens” [1,2,3]
We developed an alanine to isoleucine mutagenesis strategy in the DsbA signal sequence (DsbAss) to analyze its impact on the translocation of the targeted protein, i.e., recombinant ovine growth hormone
The current study aimed to discover the structural insights of signal peptide (DsbAss) conformation inside the fifty-four homolog (Ffh)-binding groove by using molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations
Summary
Almost all previously treatable infections have developed antibiotic resistance, leading to “multidrug-resistant pathogens” [1,2,3] This alarming situation needs a rapid solution as mortality rates by common infections caused by these multidrug-resistant pathogens will soon be much higher than those of the most feared disease, cancer [4]. From studies on a wide range of bacteria, the importance of “disulfide bond (Dsb) oxidative folding machinery” has emerged as the main area of interest [7,8]. The “Dsb” protein family has been comprehensively characterized, and type “DsbA” was considered as strongly oxidizing [10], and significantly important in the initiation process [7,11,12]
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