The N-terminal sequence after residue 247 plays an important role in
structure and function of Lon protease from Brevibacillus thermoruber WR-249
Jiun-Ly Chir
a, Jiahn-Haur Liao
a, Yu-Ching Lin
b, Shih-Hsiung Wu
a,b,*a
Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Sec. 2, Nankang, Taipei 115, Taiwan
bInstitute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan
a r t i c l e
i n f o
Article history: Received 16 March 2009 Available online 24 March 2009
Keywords: Lon protease
ATP-dependent protease Gel filtration chromatography Brevibacillus thermoruber
a b s t r a c t
Previous studies on the N-terminal domain of Lon proteases have not clearly identified its function. Here we constructed randomly chosen N-terminal-truncated mutants of the Lon protease from Brevibacillus thermoruber WR-249 to elucidate the structure–function relationship of this domain. Mutants lacking amino acids from 1 to 247 of N terminus retained significant peptidase and ATPase activities, but lost 90% of protease activity. Further truncation of the protein resulted in the loss of all three activities. Mutants lacking amino acids 246–259 or 248–256 also lost all activities and quaternary structure. Our results indicated that amino acids 248–256 (SEVDELRAQ) are important for the full function of the Lon protease.
Ó 2009 Elsevier Inc. All rights reserved.
ATP-dependent protease Lon, consists of a variable N-terminal domain (N domain), a central ATPase domain (A domain), and a C-terminal protease domain (P domain) on a single polypeptide. The Lon protease was the first ATP-dependent protease from Esch-erichia coli (Ec-Lon) and most Lon studies have largely focused on all aspects of the Ec-Lon[1]. The sizes of each domain of various Lon proteases have been compared[2], and the domains of Ec-Lon have been identified[3–5]. Most studies of Lon function and structure have focused on the A and P domain[6–10]. N domain has not fully revealed its function due to few studies.
Previous reports indicated that the N domain is related to or in-volved in binding and recognition of substrate proteins[11–13]. Our previous study showed that the N-terminal domain is essential for oligomerization; failure of oligomerization can lead to the inac-tivation of the A and P domain functions[14]. The N domain is also thought to function in the discrimination and recognition of sub-strates and in domain–domain interactions [3,14,15]. Recently, the three-dimensional structure of the N-terminus of Ec-Lon was partially resolved[11], but the three-dimensional structure of a full-length Lon protease is lacking. On the basis of the partial struc-ture, N domain appears to be a general protein- and polypeptide-interaction domain. In the present study, we investigated the role of the N domain of Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) with constructed N-terminal truncated proteins, and identified nine amino acids (SEVDELRAQ; residues 248–256)
in the N domain is essential for the full function and structure of Bt-Lon.
Materials and methods
Bacterial strains, enzymes, and chemicals. ECOS 101 [F (U80d lacZDM15)D(lacZYA-argF)U169 hsdR17(rkrk+) recA1 endA1 relA1
deoRk], used in cloning experiments, and ECOS-21 [E. coli B F dcm,
ompT, hsdS (rBmB), galk (DE3)], used for expression, were
pur-chased from ECOS (Taiwan). The substrates used for assays of ATP-ase, peptidATP-ase, and protease were purchased from Sigma: adenosine 50-triphosphate (ATP), fluorogenic peptides,
glutaryl-Ala-Ala-Phe-methoxynaphthylamide (Glt-AAF-MNA), and fluores-cein isothiocyanate (FITC)-
a
-casein type I. The protein molecular weight standards for analytical gel filtration were purchased from Amersham Biosciences (GE Healthcare, USA). All other chemicals were analytical grade.Construction of Bt-Lon mutants, protein production, purification, and activity assays. The mutants were constructed and then checked by DNA sequencing (see Supporting information). The genes encoding Bt-Lon and mutant proteins were over-expressed in E. coli strain BL-21. The proteins were purified as described pre-viously[16]. Protein concentrations were determined using the Bradford method (Bio-Rad) and bovine serum albumin as the stan-dard. The homogeneity of the purified proteins was analyzed by SDS–PAGE. ATPase, peptidase, and protease activities of Bt-Lon and mutant proteins were assayed as described previously[14]. The activities of Bt-Lon were considered as 100% activity; results given are the average of three experiments.
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.109
*Corresponding author. Address: Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Sec. 2, Nankang, Taipei 115, Taiwan. Fax: +886 2 2653 9142.
E-mail address:[email protected](S.-H. Wu).
Biochemical and Biophysical Research Communications 382 (2009) 762–765
Contents lists available atScienceDirect
Biochemical and Biophysical Research Communications
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y b b r cCircular dichroism (CD) spectra of Bt-Lon,D246–259 andD248– 256 mutants. CD spectra were recorded on a Jasco J-715 spectropo-larimeter with 0.1 cm light path at protein concentrations of 1
l
M (0.089 mg ml1) in 0.4 mM NaH2PO4, 0.08 mM KH2PO4, 0.12 mM
KCl, 0.004 M NaCl, and 2% glycerol, pH 7.4. The far-UV CD spectra were the mean of three accumulations with a 1.0 nm bandwidth.
Analytical gel filtration chromatography. Purified proteins were applied to a Superose 10/300 GL column (GE Healthcare) equili-brated with 50 mM Tris–HCl (pH 8), 10 mM MgCl2, 300 mM NaCl,
400 mM imidazole, and 10% glycerol; proteins were eluted at a flow rate of 0.3 ml min1. Blue dextran 2000 was used to
deter-mine the column void volume (V0) and elution volumes (Ve) of
the standards (GE Healthcare); the elution volumes were deter-mined from the volume of the eluent at the point of application to the center of the elution peak detected at 280 nm. Kavvalues
for the standards were calculated as (Ve V0)/(Vt V0), where Vt
is the total bead volume of the column. The standard curve was plotted as the logarithm of the molecular weight against the Kav
of the standard proteins. The corresponding Kavfor Bt-Lon and
the mutant proteinsD246–259 andD248–256 was calculated to determine their molecular weight from the calibration curve.
Results and discussion
The N domain of Bt-Lon dominates the enzyme’s oligomeric state and influences ATPase, peptidase and protease activity
Previous sequence alignments of Lon proteases from various bacterial species have shown that this N domain is poorly con-served and of variable length[2,16], yet this domain is the most stable part of the Lon protease (E. coli), as shown by both limited proteolysis and autolysis[3–5]. For example, limited trypsin diges-tion of Mycobacterium smegmatis Lon (Ms-Lon) yields a stable N-terminal fragment, which indicates that this fragment of approxi-mately 220 residues forms an independently folded domain[13]. Mutants of Ms-Lon lacking 90–225 N-terminal residues exhibit low-level peptidase activity, and a mutant lacking 277 N-terminal residues displays neither peptidase nor ATPase activity[13]. The previous studies have suggested that the N-terminal domain of Lon plays an important role in the full function of this protease.
In our earlier study, we showed that the N-terminal 316 amino acids of Bt-Lon play a role in oligomerization of the quaternary structure[14]. This fragment is, however, too large to determine which motif or which sequences form the key domain for the full function of Lon protease. In the present study, we therefore succes-sively truncated the N-terminus of Bt-Lon (D1–129,D1–175,D1– 219,D1–232,D1–247,D1–256,D1–265, andD1–279) and mea-sured the ATPase, peptidase, and protease activities of the mutant
proteins relative to those of full-length Bt-Lon (Fig. 1andTable 1). Mutant proteins with deletions up to residue 247 had only <10% of the protease activity of full-length Bt-Lon, but the ATPase and pep-tidase activities of these mutant proteins were affected to a lesser extent. However, the larger deletions (D1–256,D1–265, andD1– 279) led to loss of all activities. The quaternary structures of these mutant proteins also changed (dimer–trimer) as determined from analytical ultracentrifugation as compared with Bt-Lon (hexamer) (seeSupporting information, Table S1). The results demonstrated that (1) region 1–247 of Bt-Lon probably is less involved in the ATPase and peptidase activities and these two activities can toler-ate drastic changes in the structure, and (2) Bt-Lon requires resi-dues 1–247 as the discriminator for specific substrates or as the initiator site to hold nonspecific substrates (
a
-casein as substrate in this case) with an affinity strong enough or a period long enough to activate ATP hydrolysis, which would cause a conformational change in Bt-Lon and make the active site accessible for subse-quent hydrolysis of the protein substrate, a model proposed for Lon proteolysis by Goldberg and Waxman [17]. Our results on the protease activities of the truncated mutants of Bt-Lon are con-sistent with results reported for truncated mutants of Ms-Lon[13]. Although Ec-Lon mutant obtained by limited proteolytic digestion, lacking the N domain but still containing the A and P domain (235– 784 residues), has significant protease activity against b-casein un-der several special conditions and in the prolonged incubation (about 16–24 h), AP fragment of Ec-Lon has no ability to hydrolyze b-casein under standard reaction conditions (such as in the pres-ence of nucleotides) and in the relatively short time of incubation (about 2 h)[3].The N-terminal sequence after residue 247 plays an important role in structure and function
Considering the above results and a postulation[11] derived from the crystal structure of the N-terminus of Ec-Lon, the N do-main of Bt-Lon can be regarded as a general protein/polypeptide interaction domain that interacts with proteins targeted for degra-dation, i.e., the discriminator/initiator site is located at the N-ter-minus of Bt-Lon. Although Smith et al. [18] have shown that a sensor and substrate discrimination domain (SSD) is located in the
a
-domain of Ec-Lon and Ec-Clp family, we cannot rule out the possibility that the N domain of Bt-Lon plays a role in substrate discrimination. Interestingly, Bt-Lon mutants D1–256, D1–265, and D1–279 lacked ATPase, peptidase, and protease activities; the loss of activities by these constructs, in comparison with the forms lacking only the N-terminal sequences from 1–129 up to 1–247 confirms the importance of the fragment 248–256 for the activity of the enzyme. In addition, previous results indicated that coiled-coil regions in the N-terminus (residues 184–226 and 238–Fig. 1. N-terminal amino acid sequences of Bt-Lon (Accession No. AY197372) and each N-terminal truncated mutant was constructed from the beginning of Met (d) to the amino acid position pointed out by the symbol (N) and numbered above each of the symbols.
279) of Bt-Lon[16]might participate in the binding of Lon to pro-teins. Residues 248–256 are therefore probably part of the coiled-coil conformation, and these amino acids are probably involved in protein–protein interaction.
We tested this hypothesis by constructing two deletion mutants (D246–259, D248–256) within this sequence and assayed their activities (Table 1). Mutants D246–259 and D248–256 lost all activities. Moreover, these deletions slightly affected the protein conformation, as indicated by the CD spectra (Fig. 2), although the CD spectra revealed ellipticities at 222 nm, indicating that a he-lix was still the major component. To gain further insights into the differences in the structure of Bt-Lon and that of the two deletion mutants, we compared their quaternary structures using gel filtra-tion chromatography (Fig. 3) and sedimentation velocity. The molecular weights were estimated to be 550 kDa (hexamer) for Bt-Lon and 180–190 kDa (dimer) for mutants D246–259 and D248–256. As compared with previous measured data [14], the predominant S value of Bt-Lon is shifted from 15S to 8.6S due to a different working condition (10% glycerol and 400 mM imidazole was added to buffer at this measurement for its stability and solu-bility in this case, seeSupporting information, Fig. S1). In spite of the difference in working condition, the tendency in Mwbased on
size exclusion were consistent with those of sedimentation veloc-ity, indicating the predominant peak of Bt-Lon was 8.6 S whereas it shifted to the interval of 1.5–4.0 S for two deletion mutants. These results indicated that deleting residues 248–256 of Bt-Lon not only
changes its quaternary structure but also drastically affects all functions. These amino acids (SEVDELRAQ) therefore play a key role in the function and structure in subunit–subunit interactions. It has been reported that conformational changes in Lon holo-enzyme induced by nucleotides or protein substrates modulate the functional activities through domain–domain interactions
[15,19,20]. The domain–domain interactions of the Bt-Lon holo-enzyme might be blocked by the deletion of 248-SEVDELRAQ-256. Although the partial structure of an N-terminal subdomain (residues 1–119) of Ec-Lon has been reported[11], the structure of residues 248–256 of Bt-Lon is unknown. We therefore cannot rule out the possibility that residues 248–256 play a role in do-main–domain interactions within one subunit of Bt-Lon. How-ever, our data revealed that these residues are crucial for the function and quaternary structure. The relationship between the quaternary structure and protease activity needs to be inves-tigated further.
Acknowledgments
This research is supported by the National Science Council, Tai-pei, Taiwan, Grant Nos. NSC 95-2311-B-001-033, NSC 96-2311-B-001-010.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, atdoi:10.1016/j.bbrc.2009.03.109.
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Relative ATPase, peptidase and protease activities of Bt-Lon and mutant proteins. The activities of Bt-Lon were set to 100%. Activities were measured in 50 mM Tris–HCl, pH 8, 1 mM ATP, and 10 mM MgCl2at 50 °C.
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Fig. 2. Circular dichroism spectra of full-length Bt-Lon (h) and the deletion mutants D246–259 (s) and D248–256 (D) from 200 to 250 nm. Spectra were recorded on a Jasco-715 instrument at 25 °C. The far-UV CD spectra were the mean of three accumulations with a 0.1 cm light-path cell.
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