National Laboratory of Membrane Protein Structure (MPS), Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Korea
National Lab. of Membrane Protein Structure (MPS), Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Korea
Helicobacter pylori is a gram-negative and spiral-shaped bacteria that lives in the stomach and is associated with many serious gastric problems, ranging from gastritis to gastric carcinoma or lymphoma. It has a unique way of adapting in the harsh, acidic environment of the human stomach. Because of its importance as a human pathogen, our interest in its biology and evolution, and the value of proteomics for drug discovery and vaccine development, we determined the solution structure of HP1242, one of the proteins from H. pylori by nuclear magnetic resonance (NMR).1–4
The HP1242 gene of H. pylori encodes a 76-residue conserved hypothetical protein from H. pylori strain 26695 with a molecular weight of 9111 Da and a calculated isoelectric point of 6.1. Based on the sequence homology, this protein is classified as the DUF (Domain of Unknown Function) 465 family (pfam, http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cig), which has an unknown function. These family members are found in several bacterial proteins, and also in the heavy chain of eukaryotic myosin and kinesin, which are predicted to form coiled coil structures.
Materials and Methods.
The H. pylori genomic DNA was purchased from American Type Culture Collection (ATCC). Oligonucleotides and Taq DNA polymerase PCR premix were purchased from Bioneer Co. (Daejeon, Korea). Restriction endonucleases were purchased from Promega (Madison, WI), and New England Biolabs (Ipswich, MA). T4 DNA ligase was purchased from Promega. Escherichia coli BL21 (DE3) codon+ and pET-21a were purchased from Novagen Inc. (Darmstadt, Germany). 13C-glucose and 15N-NH4Cl were purchased from Cambridge Isotope (Andover, MA). All reagents except for the list mentioned above were either analytical or biotechnological grade. Column resins, Ni2+-agarose column (His-bind Resin) and DEAE-Sepharose column were purchased from Amersham Pharmacia Biotech Inc. (Uppsala, Sweden).
For the expression of HP1242, pET-21a was used as a vector system. The gene product has six additional histidines at the C-terminal to facilitate purification by Ni2+-agarose column (His-bind Resin). The ORF of HP1242 was amplified by PCR with appropriate primers and inserted between NdeI and XhoI cleavage sites. The constructed plasmid was transformed into competent cell, E. coli BL21 (DE3) codon+.
15N and 13C-uniformly labeled protein was prepared by growing the cells in the isotope-supplemented in M9 medium at 37°C. The protein was purified using a Ni2+-agarose column (His-bind Resin) and DEAE-Sepharose column. The NMR sample was prepared in 90% H2O/10% D2O containing 50 mM NaH2PO4/Na2HPO4 (pH 6.8), 100 mM NaCl, and 1 mM ethylenediaminetetraacetic acid.
All NMR measurements were performed at 303 K on a Bruker Avance 600-MHz NMR spectrometer. The experiments recorded on 15N-labelled sample were two-dimensional (2D) 15N-HSQC, 3D 15N-NOESY-HSQC, 3D 15N-TOCSY-HSQC, and 3D 15N-HNHA. The experiments recorded on the 15N/13C-labelled sample were 13C-NOESY-HSQC, HNCA, HNCO, HN(CO)CA, HNCACB, CBCA(CO)NH, HNCACO, HCCH-TOCSY, and CCCONH. The 3D 15N-TOCSY-HSQC used a mixing time of 50 ms. The 3D 15N-NOESY-HSQC used mixing times of 100 and 150 ms and the 3D 13C-NOESY-HSQC used a mixing time of 100 ms.
Chemical Shift Assignment and Structure Calculation.
The proton chemical shift was referenced to the methyl signal of 2,2-dimethylsilapentane-sulfonic acid (DSS) externally. The 15N and 13C chemical shifts were referenced indirectly to DSS.5 All spectra were processed using the nmrPipe/nmrDraw software,6 and were analyzed using the program NMRView.7
The additional eight residues (LEHHHHHH) at the C-terminus were not used in the structure calculation, because these residues are essentially unstructured. The distance restraints for the structure calculation were collected from 3D 15N-NOESY-HSQC and 13C-NOESY-HSQC by manual and automatic assignments for which CYANA 2.0 was used.8 Dihedral angle restraints were calculated from chemical shifts using Torsion Angle Likelihood Obtained from Shift and sequence similarity (TALOS)9 and overall secondary structure was predicted from Chemical Shift Index (CSI)10 and NOE pattern. Structure calculations were performed through standard annealing and torsion-angled dynamics using the program CNS 1.1.11 The program MOLMOL12 was used to visualize the result of 20 energy-minimized conformers. Analysis of the quality of the final structure was accomplished using PROCHECK-NMR13 and Aqua.14
Results and Discussion.
Characteristic parameters for the NMR structure calculation of HP1242 are summarized in Table I, and the visual display of the structure is shown in Figure 1. The atomic coordinates of the final structure have been deposited in the Protein Data Bank (PDB) with the code 1ZHC. HP1242 adopts a full helical structure, which is composed of three α-helices. These correspond to residues 6–14 (αI), 18–38 (αII), and 43–75 (αIII). The length of αIII is very long, which is unexpected because the prediction of secondary structure using amino acids sequence showed that the residues that belong to αIII form two helices (data not shown). The overall structure of HP1242 represents a coiled-coil-like conformation.
Table I. Structural Statistics for the 20 Energy-Minimized Conformers of HP1242
The default parameters and force constants of protein-allhdg.param. and anneal.inp in CNS 1.1 were used for structure calculation.
This is determined by Procheck.
A. Experimental constraints
NOE constraints total
Short-range (|i−j| ≤1)
Medium-range (1<|i−j| <5)
Long-range (|i−j| ≥5)
B. RMSD to the mean structure (Å) for residues 7–70
As the result of a structural homology search using DALI (http://www.ebi.ac.uk/dali/fssp/), the solution structure of HP1242 has a homology with a variety of proteins. The proteins that have a higher Z score of DALI analysis than 5.5 include rop protein [transcription regulation; PDB code 1nkd, Z score = 6.3, root-mean-square deviation (RMSD) = 2.4, Fig. 1E], arfaptin 2 fragment (signaling protein; PDB code 1i4d, Z score = 6.0, RMSD = 2.3, Fig. 1F), sensory rhodopsin ii fragment (membrane protein complex; PDB code 1h2s, Z score =5.8, RMSD = 3.8), ampiphysin fragment (endocytosis; PDB code 1uru, Z score = 5.7, RMSD = 2.7), and colicin Ia biological unit (transmembrane protein; PDB code 1cii, Z score = 5.6, RMSD = 1.8).
This result indicates that the function of HP1242 cannot be evaluated by only structural comparison. It is very probable that HP1242 is not a functional protein by itself but a subunit of a complex system.