Structure of the Month: March 2009 [see all]
Structure determination of the cancer-associated Mycoplasma hyorhinis protein Mh-p37
Sippel KH, Robbins AH, Reutzel R, Domsic J, Boehlein SK, Govindasamy L, Agbandje-McKenna M, Rosser CJ, McKenna R.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA.
The Mycoplasma hyorhinis protein Cypl (formerly p37) was originally identified as a 37 kDa protein that induced invasiveness in mouse sarcoma cells (9, 10). Since Cypl was recognized, several studies have indicated a correlation between the bacteria M. hyorhinis and gastric carcinoma, colon carcinoma, esophageal cancer, and lung cancer (4, 6). Though the function of Cypl is unknown, the operon it resides in bears some sequence similarity to a periplasmic binding-protein-dependant transport system found in Gram-negative bacteria. It has been theorized to function analogously as an extracytoplasmic binding lipoprotein, though this had never tested experimentally (2, 3, 7).
Crystallization and preliminary X-ray data of Cypl were previously reported. However low (~15%) sequence identity to previously solved structures made molecular replacement phasing methods impossible (5). Additionally, the low pH of the crystallization conditions (pH 3) interfered with the binding of heavy atoms, and thus left the structure unsolved for six years, despite much effort. The recent report of a new heavy atom derivative, 5-amino-2,4,6-triiodoisophthalic acid (I3C) presented a straight forward and inexpensive opportunity to phase the structure of Cypl (1). I3C proved to be uniquely suited to solve the Cypl structure as it allowed for the binding of the heavy atom compound, at low pH. An "in-house" room temperature X-ray diffraction data collection from a single Cypl, quick soaked in I3C, was sufficient to identify the positions of five iodine positions by SHELXD. In addition to phasing the molecule, the ligand thiamine pyrophosphate (TPP) was identified in a binding cleft of Cypl, supporting the theory that the protein is a extracytoplasmic binding protein (7, 8).
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Figure 1: Cypl Cα trace colored from low to high (blue to red) temperature factors. Ligands are marked with red circles |
All X-ray diffraction data were collected using an in-house R-AXIS IV++ image plate system with Osmic mirrors and a Rigaku RU-H3R Cu rotating anode operating at 50 kV and 100 mA. The data was collected with a detector-crystal distance of 100 mm, using oscillation steps of 1° with 8 min exposures per image. One native crystal with 170 frames and one IC3 soaked crystal with 200 frames, both with 1.9 Å resolution were collected.
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Figure 2: Thiamine Pyrophosphate binding site. A) 2|Fo-Fc| electron density of TPP. Contoured at 2σ. B) Binding interactions of TPP within the Cypl cleft. Waters colored in red. Amino acids are as labeled. Atoms of TPP are as labeled in Figure 2A. |
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Figure 3: 5-amino-2,4,6-triiodoisophthalic acid binding sites. A and B) site I; A) 2|Fo-Fc| electron density contoured to 1.7σ. B) Binding interactions of I3C. C and D) site II; C) 2|Fo-Fc| electron density contoured to 1.7σ. D) Binding interactions of I3C. Waters colored in red. Amino acids are as labeled. Atom numbering is as given in Figure 3A. |
This work was performed in the laboratory of Robert McKenna at the Center for Structural Biology, University of Florida, Gainesville, FL.
Data collection details
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Sample |
Native |
IC3 soak |
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PDB ID |
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Space group |
P21 |
P21 |
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Unit cell |
a =49.5 Å, b = 67.6 Å, c = 59.8 Å |
a =50.1 Å, b = 69.2 Å, c = 60.3 Å |
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Radiation |
Cu Kα |
Cu Kα |
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Generator |
RUH3R |
RUH3R |
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Optic |
Blue |
Blue |
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Detector |
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Crystal-to-detector distance |
100 mm |
100 mm |
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Exposure time per frame |
8 min |
8 min |
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Oscillation width |
1° |
1° |
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Number of frames |
170 |
200 |
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Data Processing |
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Resolution range |
40.0 – 1.9 Å |
50.0 – 1.9 Å |