Mol. Cells, Vol. 20, No. 3, pp. 442-445
Communication
Molecules and Cells KSMCB 2005
Backbone 1H, 15N, and 13C Resonance Assignments and Secondary-Structure of Conserved Hypothetical Protein HP0894 from Helicobacter pylori Kyung-Doo Han, Sung-Jean Park, and Bong-Jin Lee* National Laboratory of Membrane Protein Structure (MPS), Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea. (Received September 23, 2005; Accepted September 30, 2005)
HP0894 (SwissProt/TrEMBL ID O25554) is an 88residue conserved hypothetical protein from Helicobacter pylori strain 26695 with a calculated pI of 8.5 and a molecular weight of 10.38 kDa. Proteins with sequence similarity to HP0894 exist in Vibrio choierae, Enterococcus faecalis, Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli O157, etc. Here we report the sequencespecific backbone resonance assignments of HP0894. About 97.5% (418/429) of the HN, N, CO, Cα, Cβ resonances of the 88 residues of HP0894 were assigned. On the basis of these assignments, three helical regions and four strand regions were identified using the CSI program. This study is a prerequisite for calculating the solution structure of HP0894, and studying its interaction with its substrates, if any, and/or with other proteins. Keywords: Backbone Resonance Assignment; Helicobacter pylori; HP0894; Hypothetical Protein; NMR; Unknown Protein.
Introduction Helicobacter pylori is a spiral-shaped Gram-negative bacterium and human pathogen that infects approximately half the world’s population. It has a unique capacity to adapt to the extreme acidic environment of the stomach and to colonize the epithelium of the stomach chronically (Marshall et al., 1984; Sachs et al., 2003). It is responsible for diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue * To whom correspondence should be addressed. Tel: 82-2-880-7869; Fax: 82-2-872-3632 E-mail:
[email protected]
(MALT) lymphoma and gastric cancer. Gastric cancer is the most frequently diagnosed and the most common cause of cancer-related deaths in Korea (Lee et al., 2003; Peek and Blaser, 2002; Rothenbacher and Brenner, 2003; Wotherspoon et al., 1993). The genomes of two prototype strains (strains 26695 and J99) of H. pylori have been sequenced, and 1,590 open reading frames (ORFs) were identified on the chromosome of strain 26695. The functions of more than 30% of these proteins remain unknown (Alm et al., 1999; Tomb et al., 1997), and their elucidation could lead to the identification of new drug targets. HP0894 (SwissProt/TrEMBL ID O25554) is an 88residue, conserved hypothetical protein from Helicobacter pylori strain 26695 with a calculated pI of 8.5 and a molecular weight of 10.38 kDa. According to the TIGR CMR database (www.tigr.org), proteins with sequence similarity to HP0894 exist in Vibrio choierae EL Tor N16961 (VCA0323, identity: 46.1%, similarity:64.0%), Enterococcus faecalis V583 (EF0513, i: 45.6%, s: 66.7%), Campylobacter jejuni RM1221 (CJE1103, i: 44.3%, s: 65.9%), Streptococcus pneumoniae R6 (spr0253, i: 45.6%, s: 66.7%), Haemophilus influenzae KW20Rd (HI0711, i: 38.0%, s: 55.4%), Escherichia coli O157:H7EDL933 (spr0253, i: 38.6%, s: 61.4%), etc. A single iteration PSIBLAST (Altschul et al., 1997) search against the nonredundant protein database, using default parameters, identified a 57 homologues with E -values better than threshold, most of which are annotated as hypothetical or uncharacterized proteins. A search with BLAST for a conserved domain (Marchler-Bauer et al., 2004) showed that HP0894 contains a DUF332 (Domain of Unknown Function) domain, equivalent to COG 3041 in the National Center for Biotechnology Information Database of Clusters of Orthologous Groups. But, in the Pfam (Bateman et al., 2002) database, HP0894 belongs to a family of proteins of the plasmid stabilization system (PF05016;
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Table 1. Chemical shifts of HN, 15N, 13CO, 13Cα and 13Cβ of HP0894. All the chemical shifts are referenced relative to the frequency of the methyl proton resonance of DSS (unit: ppm). Residue
HN
N
CO
CA
CB
Residue
HN
N
CO
CA
CB
1
MET
ND
ND
173.19
61.25
30.28
45
GLN
8.30
117.29
174.85
56.79
27.34
2
LEU
8.80
123.45
177.61
55.74
43.44
46
ASP
9.03
121.24
177.61
55.63
44.69
3
LYS
8.69
123.60
174.69
56.97
33.76
47
HIS
9.24
120.83
171.65
55.45
30.79
4
LEU
8.58
123.69
175.61
54.45
44.76
48
ALA
8.74
122.61
177.93
53.48
19.46
5
ASN
9.14
126.82
173.16
52.11
40.16
49
LEU
8.19
121.46
176.84
54.57
43.07
6
LEU
8.49
124.53
176.40
54.32
41.63
50
LYS
8.46
119.25
177.18
55.10
35.75
7
LYS
7.77
123.90
178.59
55.46
32.13
51
GLY
8.69
108.48
176.97
46.99
8
LYS
9.17
124.78
178.86
60.33
31.92
52
LYS
9.02
126.45
176.07
57.86
31.64
9
SER
8.32
111.69
175.71
61.05
62.33
53
TRP
7.61
117.95
175.61
57.43
31.94
10
PHE
6.99
124.63
176.09
61.78
39.62
54
LYS
7.14
121.08
ND
56.18
32.33
11
GLN
8.04
119.47
177.85
59.38
28.98
55
PRO
174.47
64.08
31.40
12
LYS
8.06
117.81
179.55
59.51
32.41
56
PHE
8.91
122.00
175.01
59.13
39.45
13
ASP
7.54
121.97
178.70
57.21
40.93
57
ARG
9.13
120.25
173.03
55.29
33.61
14
PHE
8.81
121.44
176.45
60.56
39.13
58
GLU
8.54
118.05
173.83
53.64
35.25
15
ASP
8.54
118.88
178.97
57.72
40.47
59
CYS
9.07
117.57
172.98
56.39
31.18
16
LYS
7.38
119.03
179.15
59.73
32.26
60
HIS
9.99
120.55
174.46
55.75
29.29
17
LEU
7.65
121.12
180.59
58.51
40.28
61
ILE
8.99
124.14
176.00
61.77
37.01
18
LEU
8.65
121.68
181.41
58.61
41.90
62
LYS
8.49
116.70
ND
54.19
33.61
19
LEU
7.69
120.35
177.86
57.61
41.98
63
PRO
ND
67.12
31.63
20
ASN
7.44
116.01
175.67
53.13
39.91
64
ASP
8.60
114.01
ND
ND
ND
21
GLY
7.80
107.17
174.27
46.15
65
VAL
7.54
117.54
172.40
64.13
33.71
22
PHE
7.79
122.17
173.30
58.81
42.09
66
LEU
9.36
130.25
174.74
53.73
42.83
23
ASP
7.81
127.60
175.13
52.82
42.02
67
LEU
8.95
122.32
175.03
53.73
45.54
24
ASP
8.48
124.11
176.37
54.02
40.85
68
VAL
8.61
126.47
175.34
61.31
33.57
25
SER
8.38
117.39
177.16
63.62
62.60
69
LYS
9.06
125.79
185.03
56.30
43.17
26
VAL
8.02
123.39
177.71
66.34
31.58
70
LEU
9.25
120.65
174.33
54.52
47.88
27
LEU
8.16
121.18
177.25
58.57
41.81
71
VAL
8.85
126.27
175.52
62.35
32.41
28
ASN
8.48
115.50
178.35
56.41
37.59
72
LYS
8.89
129.78
175.17
55.52
35.24
29
GLU
7.56
122.17
179.10
59.68
29.32
73
ASP
9.18
126.49
ND
57.06
39.66
30
VAL
8.38
123.69
177.36
66.92
31.64
74
ASP
8.47
118.63
174.68
54.18
40.56
31
ILE
8.58
118.67
177.38
66.40
38.10
75
GLU
8.07
118.40
173.24
55.35
34.35
32
LEU
7.61
119.57
178.15
58.78
42.10
76
LEU
8.96
124.21
173.37
53.88
43.38
33
THR
7.82
116.62
176.30
68.15
69.12
77
ILE
9.64
128.35
175.76
60.61
39.68
34
LEU
8.35
119.62
182.22
57.86
43.85
78
LEU
8.39
125.19
174.91
53.94
40.65
35
ARG
8.78
118.21
177.76
59.90
30.96
79
LEU
8.63
122.45
176.63
57.65
45.74
36
LYS
7.74
118.36
175.30
57.23
32.93
80
ARG
7.78
113.18
174.15
52.80
36.58
37
LYS
8.02
114.79
175.55
57.58
28.88
81
LEU
9.80
124.22
174.59
53.71
46.62
38
GLU
7.47
119.24
ND
53.49
30.65
82
GLY
8.90
111.27
171.16
45.94
177.32
62.36
32.05
83
SER
8.83
117.24
175.09
57.61
65.47
177.81
54.06
43.29
84
HIS
9.62
121.18
177.38
61.63
29.33 62.91
39
PRO
40
LEU
8.39
121.76
41
ASP
8.90
123.32
42
PRO
43
GLN
9.43
44
PHE
7.81
* ND, not detected.
ND
53.93
42.02
85
SER
8.70
112.38
176.23
61.28
178.90
65.77
32.37
86
GLU
7.51
120.70
177.70
58.29
30.02
118.60
176.98
58.36
27.81
87
LEU
7.67
118.77
178.04
57.34
41.85
116.68
174.96
59.72
38.86
88
PHE
7.73
113.99
175.98
57.73
39.44
444
NMR Study on HP0894
Fig. 1. Consensus CSI results and secondary structure of HP0894 on the basis of assigned backbone resonances.
This family also encompasses RelE/ParE.). The exact molecular function of these proteins is not known. Here we report the sequence-specific backbone resonance assignments and secondary-structure of HP0894. This study is a prerequisite for calculating its solution structure and for structural study of its interaction with substrates, if any, and /or with other proteins.
Materials and Methods HP0894 was amplified by the polymerase chain reaction with specific primers using H. pylori genomic DNA as template. The amplified DNA fragment was cloned into plasmid pET21a (Novagen) and the recombinant plasmid was transformed into Escherichia coli BL21(DE3) for large-scale protein production. The recombinant protein contains eight non-native residues at its C-terminus (LEHHHHHH) that facilitate protein purification. Uniformly 15N- and 15N/13C-labeled protein was prepared by growing bacteria in M9 medium with 15NH4Cl and 13C6-glucose as nitrogen and carbon sources. The protein was purified using a Ni2+-affinity column (Chelating Sepharose Fast Flow Resin, Pharmacia). All NMR samples were dissolved in 90% H2O/10% D2O containing ~1 mM 15N- and 15N/13C-labeled protein in 20 mM NaH2PO4/Na2HPO4 (pH 5.0), 500 mM NaCl, 0.1 mM DTT and 0.1 mM EDTA. NMR spectra were acquired on Bruker AVANCE 500 and AVANCE 600 (equipped with a cryoprobe) spectrometers at 303 K. Spectra were processed using NMRPipe/NMRDraw (Delaglio et al., 1995) and analyzed with NMRView. 1H-15N HSQC, HNCO, HNCA, HN(CO)CA (Kay et al., 1990), HNCACB (Wittekind et al., 1993), HN(CO)CACB (Grzesiek et al., 1992), 3D 15 N-separated NOESY-HSQC (Ikura et al., 1990), 3D and 15N-separated TOCSY-HSQC (Gronenborn et al., 1989) were acquired, and assignments were made from these spectra. Chemical shifts were referenced to DSS. On the basis of resonance assignments, we performed a CSI analysis (Wishart
Fig. 2. 1H-15N HSQC spectrum of HP0894. The cross peaks in the spectrum are labeled with the assigned amino acid residues. The dotted lines represent the sidechain NHs of Gln and Asn. The additional residues originating from the vector sequence are indicated by the asterisk.
and Sykes, 1994).
Results and Discussion About 97.5% (418/429) of the HN, N, CO, Cα, Cβ resonances of HP0894 were assigned (Table 1). We were unable to detect three peaks from HNCO [(64HN, 63CO, 64N), (65HN, 64 CO, 64N) and (74HN, 73CO, 74N)], four peaks from HNCA/HNCACB [(64HN, 64Cα, 64N), (64HN, 64Cβ, 64N), (64HN, 63Cα, 64N) and (64HN, 63Cβ, 64N)], and two peaks from HN(CO)CA/HN(CO) CACB [(65HN, 64Cα, 65N) and (65HN ,65Cβ, 65N)], and therefore, could not assign the 63CO, 64CO, 64Cα, 64Cβ and 73CO resonances. Interestingly residues 63, 64, and 73 are in the hinge regions between β-strands, according to the CSI analysis, and so it seems likely that these hinge-regions have a conformation-exchangeable state. Three helical regions and four strand regions were identified from the resonance assignments using the CSI program (Fig. 1). These correspond to residues 8K19S(αI), 25S-37K(αII), 84H-87L(αIII), 4L-5N(βI), 57R62K(βII), 66L-72K(βIII), and 75E-81L (βVI). Figure 2 shows the 1H-15N HSQC spectrum with the assigned amino acid residues labeled. This data will be useful in studying interactions of HP0894 with other molecules in H. pylori.
Acknowledgments This work was supported by a grant (03PJ2-PG4-BD02-0001) from the Ministry of Health & Welfare, R.O.K. It was also supported in part by a National Research Laboratory Program (M1-0412-00-0075) and a 2005 BK21 project for Medicine, Dentistry, and Pharmacy.
Kyung-Doo Han et al.
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