- Research article
- Open Access
Differential roles of tryptophan residues in conformational stability of Porphyromonas gingivalis HmuY hemophore
© Bielecki et al.; licensee BioMed Central Ltd. 2014
- Received: 25 October 2013
- Accepted: 3 February 2014
- Published: 10 February 2014
We have previously shown that the P. gingivalis HmuY hemophore-like protein binds heme and scavenges heme from host hemoproteins to further deliver it to the cognate heme receptor HmuR. The aim of this study was to characterize structural features of HmuY variants in the presence and absence of heme with respect to roles of tryptophan residues in conformational stability.
HmuY possesses tryptophan residues at positions 51 and 73, which are conserved in HmuY homologs present in a variety of bacteria, and a tryptophan residue at position 161, which has been found only in HmuY identified in P. gingivalis strains. We expressed and purified the wildtype HmuY and its protein variants with single tryptophan residues replaced by alanine or tyrosine residues. All HmuY variants were subjected to thermal denaturation and fluorescence spectroscopy analyses. Replacement of the most buried W161 only moderately affects protein stability. The most profound effect of the lack of a large hydrophobic side chain in respect to thermal stability is observed for W73. Also replacement of the W51 exposed on the surface results in the greatest loss of protein stability and even the large aromatic side chain of a tyrosine residue has little potential to substitute this tryptophan residue. Heme binding leads to different exposure of the tryptophan residue at position 51 to the surface of the protein. Differences in structural stability of HmuY variants suggest the change of the tertiary structure of the protein upon heme binding.
Here we demonstrate differential roles of tryptophan residues in the protein conformational stability. We also propose different conformations of apo- and holoHmuY caused by tertiary changes which allow heme binding to the protein.
- Porphyromonas gingivalis
- Protein unfolding
Periodontitis is an infectious disease in which genetic, microbial, immunological, and environmental factors combine to influence disease risk and progression, resulting in the destruction of tooth-supporting tissues [1, 2]. There is growing evidence showing that a number of complex human diseases are caused or are influenced by periodontal diseases, including cardiovascular and respiratory diseases, diabetes mellitus, osteoporosis, and rheumatoid arthritis [3, 4]. A major etiological agent in the development and progression of chronic periodontitis is Porphyromonas gingivalis, a black-pigmented Gram-negative anaerobic bacterium .
Establishment of infection by pathogens depends on a readily available iron, and for some bacteria a heme source. However, iron and heme availability is limited in the host environment and therefore bacteria evolved sophisticated systems to acquire these compounds from host hemoproteins. Among them, Gram-negative bacteria utilize outer-membrane receptors directly or with the assistance of a hemophore [5–9]. Unlike other Gram-negative bacteria, P. gingivalis does not produce and utilize siderophores. The bacterium also lacks the majority of enzymes of the biosynthetic pathway for heme biosynthesis. Therefore, P. gingivalis must acquire heme as a sole source of iron and protoporphyrin IX. In P. gingivalis, several TonB-dependent outer-membrane receptors for heme utilization have been described, including HmuR, Tlr, IhtA, and HusB [5, 8]. The best characterized system of heme acquisition in P. gingivalis consists of proteins encoded by hmu operon, comprising the HmuR and HmuY proteins. HmuR is an outer-membrane TonB-dependent receptor involved in heme transport through the outer membrane [10–13], whereas HmuY is a membrane-associated heme-binding lipoprotein [14–16]. We reported that HmuY binds iron(III) protoporphyrin IX and iron(II) protoporphyrin IX at 1:1 molar ratio [14, 15]. Detailed characterization of the HmuY-heme complex demonstrated that heme, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment . In that report we identified histidine residues at positions 134 and 166 as potential heme ligands. Crystallographic analysis determined unique beta-fold HmuY protein structure and confirmed engagement of H134 and H166 in heme binding . We also demonstrated that HmuY binds iron(III) mesoporphyrin IX and iron(III) deuteroporphyrin IX in an analogous way to heme . The only differences observed were two forms of HmuY-iron(III) deuteroporphyrin IX complex differing by a 180° rotation of porphyrin around the α-γ-meso-carbon axis. Recently we showed that HmuY forms complexes with non-iron metalloporphyrins, though recognition of the ligand by HmuY depends on the central metal ion . Among them, Ga(III)PPIX, Co(III)PPIX, and Cu(II)PPIX exhibited antimicrobial activity against P. gingivalis . Similar to hemophores, HmuY was demonstrated to wrest heme from methemoglobin [9, 22]. Heme extraction from oxyhemoglobin was facilitated after oxidation to methemoglobin by pre-treatment with P. gingivalis R-gingipain A (HRgpA) and K-gingipain (Kgp)  or Prevotella intermedia interpain A . Heme uptake served by HmuR receptor and hemophore-like HmuY lipoprotein is novel and has been identified for the first time in P. gingivalis.
In this work we report structural features of HmuY variants and the protein’s conformational changes induced by temperature and studied by means of fluorescence spectroscopy. Studies presented in this work have been performed using the wildtype HmuY protein and its variants with single tryptophan residues replaced by alanine or tyrosine residues. Due to the widespread location of tryptophan residues in HmuY, one could expect that observation of tryptophan fluorescence spectra could help to explain local structural stability of the protein and conformational changes accompanying ligand binding.
Site-directed mutagenesis and protein purification
Primers designed and used in this study
Preparation of holoproteins
where ε280D is the theoretical extinction coefficient (http://www.expasy.org) whilst AN280 and AD280 are absorbancies of native and unfolded protein in 20 mM phosphate buffer pH 7.4, containing 20 mM NaCl and the same buffer containing 6 M guanidine hydrochloride (Gdn-HCl; MP Biomedicals), respectively. The empirical extinction coefficients are as follows: ε280 = 32.16, 27.75, 27.66, 30.82, 29.37, 28.45 mM-1 cm-1 for W51A, W73A, W161A, W51Y, W73Y, W161Y, respectively. Titration curves were analyzed using equations for a one-site binding model and Kd values determined as reported earlier  using the OriginPro 8 software (OriginPro Corporation).
Circular dichroism (CD) spectroscopy
Far-UV CD spectroscopy was carried out using a Jasco J-810 spectropolarimeter. Protein samples at 2 μM concentration in 20 mM sodium phosphate buffer, pH 7.4, were measured in a 10-mm path-length cell. The spectra were recorded over a wavelength range of 190–260 nm by signal averaging of four spectra. Scanning speed of 100 nm/min and step resolution of 1 nm were used. All spectra were baseline corrected for respective buffers.
Intrinsic tryptophan fluorescence spectroscopy
Intrinsic tryptophan fluorescence emission spectra were recorded using a Jasco FP750 spectrofluorometer. Protein samples at 4 μM concentration in 20 mM sodium phosphate buffer, pH 7.4, containing 20 mM NaCl were excited at 295 nm and emission spectra were recorded in the range of 300–450 nm, at a scanning interval of 1 nm and integration time of 1 s. All spectra were baseline corrected for respective buffers. For analysis of heme binding, 4 μM protein samples in 20 mM sodium phosphate buffer, pH 7.4, containing 20 mM NaCl were titrated with 1-μl aliquots of 1 mM hemin prepared as described previously . Fluorescence was determined at excitation at 295 nm and emission at 329 nm.
Unfolding experiments were performed according to standard protocols with modifications described previously . Changes in intrinsic tryptophan fluorescence emission spectra due to thermally induced protein unfolding were monitored at 315 nm for excitation at 295 nm using a Jasco FP750 spectrofluorometer. Protein samples at concentration of 4 μM in 20 mM sodium phosphate buffer, pH 6.5, containing 20 mM NaCl and 1 M GdnHCl  were heated (22°C–85°C) in 10-mm path-length cells with time/temperature interval of 1°C per minute.
Sypro Orange (SYPRO ORANGE protein gel stain, Invitrogen) exhibits intensive fluorescence when bound to hydrophobic residues of proteins and therefore it was used as a probe for thermal unfolding of HmuY. Samples of 40 μM proteins in 20 mM sodium phosphate buffer, pH 7.4, containing 20 mM NaCl were placed in a 96-well qPCR plate and Sypro Orange was then added according to the manufacturer’s instructions (Invitrogen). After 1-h equilibration at 25°C the plate was heated up to 80°C in an Mx3005P thermocycler (Agilent Technologies) with a rate of 0.5°C per 30 s. Fluorescence was monitored by a single photomultiplier tube (PMT) detector using a FROX filter (excitation wavelength 585 nm, emission wavelength 610 nm). Fluorescence data were transformed to yield the relative fraction of unfolded protein .
A tryptophan in a hydrophobic environment exhibits a maximum wavelength of the fluorescence emission spectra at 320–335 nm, while in a hydrophilic or exposed environment it exhibits a maximum at 340–360 nm . Therefore intrinsic fluorescence emission, by selective excitation of tryptophan residues, was used to probe local conformational fluctuations in HmuY. When excited at 295 nm, all proteins show rather wide fluorescence emission spectra. The wildtype HmuY and all but the W51Y protein variants show maxima within a wavelength range from 328 to 330 nm. However, tyrosine substitution of the apo-form of W51 results in slight blue shift of the spectrum maximum to 325 nm. It is again in good agreement with W51 being exposed to the more polar environment. Interestingly, W51A mutant has a maximum at 328 nm, which may result from structural differences of the protein variant imposed by the lack of a large aromatic side chain on the protein surface that normally would make the structure more rigid, protecting it from penetration by solvent molecules.
While W161 is the most buried tryptophan residue in the protein core its impact on fluorescence spectra of native protein is marginal. Both alanine and tyrosine W161 variants have almost identical fluorescence spectra as the wildtype HmuY (Figure 2), which implicates that W161 residue is completely quenched. This may be due to aromatic ring π stacking between W161 and proximal F194. In addition, high energy states of W161 indoile group can be discharged by formation of the hydrogen bond between side ring nitrogen of W161 and the main chain carbonyl oxygen of Y80. Similarity in spectra of apoHmuY and holoHmuY as well as of W161 variants suggest that both hydrogen bond and tryptophan and phenylalanine ring arrangements remain the same in both protein forms.
Substitution of W51 or W73 by either a tyrosine or an alanine residue results in loss of about 18% or 60% of apoHmuY fluorescence intensity (Figure 2). W51 and W73 residues are located in close proximity to each other and in holoHmuY, as can be seen in the crystal structure , W51 being the most exposed tryptohpan residue to a more hydrophilic environment on the protein surface. Therefore energy from W73 can be transferred to W51 and then quenched due to the polar environment of the latter. This also explains why fluorescence spectra of each protein variant do not sum to the spectra of the wildtype protein. Since W161 seems not to contribute to the spectra of apoHmuY, W51A, W73A, W51Y and W73Y can serve as representatives for protein variants where only fluorescence emission spectra of W73 or W51, respectively, can be observed. We assumed that spectra of W73A and W73Y simply represent fluorescence of W51 whilst spectra of W51A and W51Y correspond to fluorescence intensity of W73 when none of its energy is transferred to W51. Thus, it has been useful for observation of HmuY thermal unfolding and concluding conformational changes accompanying ligand binding.
Finally, it should be noted that HmuY may function as monomer and, especially after heme binding, may form dimers and tetramers [15, 18]. Therefore, influence of oligomerization on protein stability and heme transfer should be taken into consideration. However, in this study we assumed that under conditions used (protein concentration at 4–40 μM) HmuY is present in solutions in the form of a monomer. Such assessment was based on our preliminary, unpublished data (i.e. results from gel filtration, analytical ultracentrifugation and small angle X-ray scattering analysis). These data suggest that HmuY oligomerization may occur at higher protein and salt concentration .
Based on our data, one may expect conformational changes occurring during heme binding to HmuY and probably during heme release from HmuY. We propose that HmuY first binds free heme or preferably wrests heme bound to hemoproteins, mainly hemoglobin, and then releases it for subsequent binding to HmuR. The initial step in heme unbinding may involve disruption of only one of the two axial histidine ligands of HmuY. Our previous studies showed that single replacement of H134 or H166 by an alanine residue did not result in abolishing heme binding, which at least in part may support this hypothesis. This reversible intramolecular coordination by a histidine chain may allow heme transfer from hemoproteins to HmuY and subsequently to HmuR, similar to typical bacterial hemophores. It has been shown that the hemophore HasA from Serratia marcescens [25, 26] or Pseudomonas aeruginosa [27, 28] exhibits large rearrangements, allowing heme binding to H32 and Y75. However, a recent study demonstrated that H32 is not conserved in all secreted hemophores. For example, HasA from Yersinia pestis coordinates heme with a single Y75 and its structure in both apo- and holoform is almost identical . Thus, analysis of heme binding to HmuY is of importance in understaning heme transfer into the P. gingivalis cell.
In this study we demonstrate differential roles of tryptophan residues played in the HmuY conformational stability. We also hypothesize that the changes observed may result from different tertiary structure of apo- and holoHmuY. At least so far, we were unable to solve the three-dimensional structure of apoHmuY, allowing detailed comparison of apo- and holoforms. Therefore results gained in this study may at least in part support our hypothesis of tertiary changes allowing heme binding to P. gingivalis HmuY.
Marcin Bielecki and Halina Wojtowicz share the first authorship.
This work was supported by Wroclaw Research Center EIT + under the project “Biotechnologies and advanced medical technologies – BioMed” (POIG 01.01.02-02-003/08/00) co-financed by the European Regional Development Fund (Operational Program Innovative Economy, 1.1.2) and by Faculty of Biotechnology research project no. 1013/S/WB/2009-2011. The funders had no role in study design, data collection and analysis, or preparation of the manuscript.
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