How do the protonation states of E296 and D312 in OmpF and D299 and D315 in homologous OmpC affect protein structure and dynamics? Simulation studies

https://doi.org/10.1016/j.compbiolchem.2014.10.006Get rights and content

Highlights

  • Different protonation states of E296(D299) and D312(315) in OmpF and OmpC promote the large-­scale deviation in protein structure and dynamics.

  • Either E296(D299) or D312(315) should be charged to preserve L3 motion.

  • OmpC is more resistant to different protonation states of E296(D299) and D312(315) than OmpF.

  • The tip of L3 controls pore cavity in bothporins.

Abstract

In this study, the structural and dynamic properties of two major porins (OmpF and OmpC) in Escherichia coli are investigated using molecular dynamics (MD) simulations. Both porins have the extracellular loop L3 folded halfway through the pore to form a constriction area. The solute influx and efflux are controlled by the L3 movement. E296 and D312 in OmpF and homologous D299 and D315 in OmpC located on the barrel wall are found to play a key role in L3 gating activity. All possible charged states of both E296(D299) and D312(315) are applied in this study to observe changes in overall structure and especially L3 movement. The results show that different protonation states of both residues cause the large-scale deviations in structure and pore cavity especially in OmpF. Fully charged E296(D299) and D312(315) increase the protein flexibility significantly. Deprotonating at least one of E296(D299) and D312(315) helps to fasten L3 to the barrel wall and maintain pore size. Lacking of interactions with D312(315) can lead to the pore closure in OmpF. Comparing with OmpC, not only is OmpF less stable, but it is also more sensitive to the charge states of both E296(D299) and D312(315).

Introduction

Porins are water-filled pore-forming proteins that reside in the outer membrane of Gram-negative bacteria. They control the influx and efflux of nutrients and metabolites (Malek and Maghari, 2007, Nikaido, 2003). Especially, porins also serve as an entryway for many antibiotics (Basle et al., 2004, Alcaraz et al., 2004, Saint et al., 1996, Schulz, 2002, Kojima and Nikaido, 2013, Nestorovich et al., 2003, Todt et al., 1992, Bonvin et al., 2000). Most porins are homotrimers and are extremely stable in a wide range of pH, organic solvents, chaotropic salts, and detergents (Phale et al., 1998). The most conspicuous structural aspect of the trimeric porins is the presence of the constriction region or ‘eyelet’ located within the hourglass-shaped channel which is lined with charged residues. This feature is conserved in general diffusion porins and is found to be important physiologically (Kindal et al., 2002). One of the most studied porins is the outer membrane protein F (OmpF) from Escherichia coli. It consists of 16 antiparallel β strands connected by turns at the periplasmic side and loops at the extracellular side (Cowan et al., 1995, Cowan et al., 1992, Hoenger et al., 1993, Darden et al., 1993, Im and Roux, 2002a, Miedema et al., 2006, Varma et al., 2006). One of the loops (L3) is folded into the lumen in each monomer. This folded L3 creates a constriction area halfway through the pore Fig. 1.

In this constriction site, a strong electric field is present due to the acidic residues on L3 and the basic residues on the opposite barrel wall. This strong electric field is essential for a pore conductance. Besides, there is the so-called latching loop (L2) reaching over to the neighbouring monomers to stabilize a trimeric form.

These structural features are also conserved in the outer membrane protein C (OmpC). OmpC shares 77% sequence similarity to OmpF and it shows similar porin architecture and secondary structure element (Kumar et al., 2010). Key residues for structure and function found in OmpF are also conserved in OmpC. Despite very similar structure, OmpC is found somewhat more cation selective and has smaller pore size (Nikaido, 2003, Nikaido, 2003). Besides, OmpC is less voltage sensitive therefore it requires higher voltage for pore closure (Schulz, 2002, Saint et al., 1996). Thus, OmpC is preferentially expressed under extreme conditions (e.g., high temperature, toxic agents, or osmolarity), whereas OmpF is repressed (Forst et al., 1989). Moreover, both porins also show different characteristics in solute uptake (Saint et al., 1996), but the origin of these differences is not well understood.

OmpF have been widely studied since the 20th century. Some key residues for both trimer stability and transport function were identified. For example, a series of saltbridges, E71(from adjacent monomer)-R100 and E71-R132, was found to involve in trimer stability (the locations of these residues are shown in Fig. 6) (Liu and Delcour, 1998a, Liu et al., 2000, Phale et al., 1998, Varma et al., 2006). Moreover, the effects of constriction loop L3 on pore gating activity are also of interest (Benz et al., 1985, Cowan et al., 1992, Im and Roux, 2002b, Karshikoff et al., 1994). The movement of L3 was found to play a role in ion selectivity and pore cavity (Im and Roux, 2002b, Karshikoff et al., 1994). Based on the crystal structure (Cowan et al., 1992), The tip of L3 is expected to interact with the opposite barrel wall via hydrogen bonds involving E117 on L3 and 2 other residues E296 and D312 on the adjacent wall (see Fig. 6 for locations). Several OmpF experiments revealed that D312 and E296 on the opposite barrel wall are crucial for L3 flexibility (Basle et al., 2006, Cowan et al., 1992, Karshikoff et al., 1994, Liu and Delcour, 1998a). All key residues for both structure and function are also conserved in OmpC as seen in Fig. 1E. Like OmpF, OmpC employs L3 for gating activity. Based on key residues found for trimer stability and function, the electrostatic interactions seem to be the major driving force. Therefore, the charge states of key ionisable residues become important. Previous pKa calculations of OmpF indicated the uncommon charge states of important residues such as E296 and D312 (Karshikoff et al., 1994). Many studies suggested possible charged states of the two residues. Initially, both E296 and D312 were computationally found to be protonated at physiological pH (Im and Roux, 2002b, Karshikoff et al., 1994, Miedema et al., 2006, Alcaraz et al., 2009, Tieleman and Berendsen, 1998, Schirmer and Phale, 1999, Humphrey et al., 1996). Later, protonating either E296 or D312 and deprotonating both residues were also employed in some simulations (Varma et al., 2006, van der Straaten et al., 2002, Biro et al., 2010). Nonetheless, based on a previous patch clamp study, deprotonating D312 only becomes more reasonable (Liu and Delcour, 1998a). The patch clamp work revealed that the D315A mutation in OmpC (D315 is homologous to D312 in OmpF) resulted in an increase in the frequency of pore closures. This indicates the key role of charged D315 on tethering L3 to the opposite wall. Hence, it is thought that homologous D312 in OmpF should act in the same manner. To date, it seems that the suitable charge states of the two residues and especially clear molecular details responding to different protonation states remain obscure. So, this study aims at understanding the structural responses when all possible protonation states of E296(D299) and D312(D315) are applied in both OmpF and OmpC (the residue number in a bracket represents the homologous residue number of OmpC). The differences in structure and dynamics (especially the trimeric stability and L3 motion) affected by different protonation states of the two are investigated.

Molecular dynamics (MD) simulations have been used in a number of earlier studies to investigate the dynamic properties of OmpF. A 1 ns MD study of the trimeric OmpF revealed deviations of dynamical structure relative to the crystal structure. It also showed that L3 flexibility affected a change in pore cavity (Humphrey et al., 1996). MD simulations were also successfully used to observe the solute (such as antibiotics) passage through OmpF in comparison with experiments (Malek and Maghari, 2007, Kumar et al., 2010, Singh et al., 2013, Ziervogel and Roux, 2013, Mahendran et al., 2010). OmpF are computationally well studied whereas few computational studies on OmpC are found. Recently, the accelerated MD has revealed the antibiotic permeations through both OmpC and OmpF (Kumar et al., 2010). They observed the more extended but narrower constriction area and the more antibiotic specificity of OmpC (Kumar et al., 2010). Furthermore, MD simulations were used to study the influence of protonation states of titratable residues on the OmpF function (Im and Roux, 2002a, Im and Roux, 2002b, Varma et al., 2006, van der Straaten et al., 2003, Berendsen et al., 1984). It appears that key aspects of the protein behavior can be captured on the MD timescale. Therefore, MD simulations are employed in this study to examine the effect of different protonation states of E296 and D312 (homologous to D299 and D315 in OmpC) on trimer stability and L3 activity in both porins in atomic level. This study has no intention to predict the suitable charge states for the two key residues. It is aimed at understanding the nature of OmpF and OmpC in microscopic detail when some unusual charged residues suggested from previous studies are applied. This study may partially reflect the structural responses from both porins at different pH.

Section snippets

Simulation setup

The trimeric OmpC (2J1N, resolution 2.0 Å) and OmpF (2OMF, resolution 2.4 Å) crystal structures consisting of 346 and 340 amino acids in each monomer respectively were used in this study. The charges of E296 and D312 in OmpF and D299 and D315 in OmpC were set separately, whereas the rest ionisable residues were set as a common state at physiological pH. All possible protonation states of E296(D299) and D312(315) were set in this study. These 4 different protonation states for both porins were:

Structural properties of OmpC and OmpF

Overall, the RMSDs, calculated on alpha carbons, demonstrate that the barrel wall of OmpC is more stable than that of OmpF. Importantly, different protonation states of E296 (D299 in OmpC) and D312 (D315 in OmpC) do influence the structural stability, especially in fully charged E296(D299) and D312(D315) (NP). However, this protonation variation appears to have more impact on loop stability. The high RMSDs of NP clearly indicate the increase in protein fluctuation in both porins (Fig. 2; blue

Conclusion

Like earlier studies (Kumar et al., 2010, Dhakshnamoorthy et al., 2010, Heller and Wilson, 1981), this study can also confirm that OmpC is more structurally stable than OmpF. Its pore size is also slightly smaller than that of OmpF. However, this study focuses on the effect of ionization states of E296(D299) and D312(315) at the barrel wall on L3-wall attachment in both OmpC and OmpF. It is interesting that the different protonation states of only two ionisable residues (E296(D299) and

Acknowledgements

Financial support is granted by Kasetsart University Research and Development Institute (KURDI)(code: PT(D)86.56). Computer facilities are supported by Dr. Jitti Niramitranon, Department of computer engineering, Kasetsart University, Thailand. Many thanks to Assist. Dr. Jirasak Wong-ekkabut for his kind support.

References (46)

  • W. Im et al.

    Ions and counterions in a biological channel: A molecular dynamics simulation of OmpF porin from Escherichia coli in an explicit membrane with 1M KCl aqueous salt solution

    J. Mol. Biol.

    (2002)
  • A. Karshikoff et al.

    Electrostatic properties of two porin channels from Escherichia coli

    J. Mol. Biol.

    (1994)
  • N. Liu et al.

    Inhibitory effect of acidic pH on OmpC porin: wild-type and mutant studies

    FEBS Lett.

    (1998)
  • K. Malek et al.

    Translocation and interactions of L-arabinose in OmpF porin: a molecular dynamics study

    Biochem. Biophys. Res. Commun.

    (2007)
  • E.M. Nestorovich et al.

    Residue ionization and ion transport through OmpF channels

    Biophys. J.

    (2003)
  • N. Saint et al.

    Structural and functional characterization of OmpF porin mutants selected for larger pore size functional characterization

    J. Biol. Chem.

    (1996)
  • T. Schirmer et al.

    Brownian dynamics simulation of ion flow through porin channels

    J. Mol. Biol.

    (1999)
  • G.E. Schulz

    The structure of bacterial outer membrane proteins

    Biochim. Biophys. Acta

    (2002)
  • D.P. Tieleman et al.

    A molecular dynamics study of the pores formed by Escherichia coli OmpF porin in a fully hydrated palmitoyloleoylphosphatidylcholine bilayer

    Biophys. J.

    (1998)
  • S. Varma et al.

    The influence of amino acid protonation states on molecular dynamics simulations of the bacterial porin OmpF

    Biophys. J.

    (2006)
  • S. Varma et al.

    Ionization states of residues in OmpF and mutants: effects of dielectric constant and interactions between residues

    Biophys. J.

    (2004)
  • B.K. Ziervogel et al.

    The binding of antibiotics in OmpF porin

    Structure

    (2013)
  • A. Basle et al.

    Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF

    Protein Eng. Des. Sel.

    (2004)
  • Cited by (4)

    • Comparative analyses and molecular videography of MD simulations on WT human SOD1

      2022, Computational and Theoretical Chemistry
      Citation Excerpt :

      Recent MD simulations for SOD1 which also report methodological advancements include discrete molecular dynamics with FMO calculations [12–14], steered molecular dynamics with geometric sampling [15,16], coarse-grained molecular dynamics [17], artificial intelligence (neural network) acceleration [18], custom CHARMM parameterizations [19], and basic metadynamics [20]. Myriad MD studies both on SOD1 and other computational constructs contributed as precedent [13,21–36,36–50]. The simulations of this manuscript are high-resolution rather than extended-time in order to analyze the matrix, metadynamics, and mechanics.

    • Why do the outer membrane proteins OmpF from E. coli and OprP from P. aeruginosa prefer trimer? Simulation studies

      2016, Journal of Molecular Graphics and Modelling
      Citation Excerpt :

      Studies of trimeric OmpF revealed deviations of dynamical structure relative to the crystal structure. They also showed that L3 flexibility affected a change in pore cavity [32,33]. MD simulations were also successfully used to observe behaviour and solute (such as antibiotics) passage through OmpF in comparison with experiments [2,33–37].

    View full text