Crystal structure and functional implications of the tandem-type universal stress protein UspE from Escherichia coli
© Xu et al. 2016
Received: 17 July 2015
Accepted: 1 February 2016
Published: 11 February 2016
The universal stress proteins (USP) family member UspE is a tandem-type USP that consists of two Usp domains. The UspE expression levels of the Escherichia coli (E. coli) become elevated in response to oxidative stress and DNA damaging agents, including exposure to mitomycin C, cadmium, and hydrogen peroxide. It has been shown that UspA family members are survival factors during cellular growth arrest. The structures and functions of the UspA family members control the growth of E. coli in animal hosts. While several UspA family members have known structures, the structure of E. coli UspE remains to be elucidated.
To understand the biochemical function of UspE, we have determined the crystal structure of E. coli UspE at 3.2 Å resolution. The asymmetric unit contains two protomers related by a non-crystallographic symmetry, and each protomer contains two tandem Usp domains. The crystal structure shows that UspE is folded into a fan-shaped structure similar to that of the tandem-type Usp protein PMI1202 from Proteus mirabilis, and it has a hydrophobic cavity that binds its ligand. Structural analysis revealed that E. coli UspE has two metal ion binding sites, and isothermal titration calorimetry suggested the presence of two Cd2+ binding sites with a Kd value of 38.3–242.7 μM. Structural analysis suggested that E. coli UspE has two Cd2+ binding sites (Site I: His117, His 119; Site II: His193, His244).
The results show that the UspE structure has a hydrophobic pocket. This pocket is strongly bound to an unidentified ligand. Combined with a previous study, the ligand is probably related to an intermediate in lipid A biosynthesis. Subsequently, sequence analysis found that UspE has an ATP binding motif (Gly269- X2-Gly272-X9-Gly282-Asn) in its C-terminal domain, which was confirmed by in vitro ATPase activity monitored using Kinase-Glo® Luminescent Kinase Assay. However, the residues constituting this motif were disordered in the crystal structure, reflecting their intrinsic flexibility. ITC experiments revealed that the UspE probably has two Cd2+ binding sites. The His117, His 119, His193, and His244 residues within the β-barrel domain are necessary for Cd2+ binding to UspE protein. As mentioned above, USPs are associated with several functions, such as cadmium binding, ATPase function, and involvement in lipid A biosynthesis by some unknown way.
KeywordsUspE UspA superfamily Tandem-type USP
The universal stress proteins (USP) superfamily is a group of conserved proteins that play an important role in E. coli. USPs’ expression levels become elevated in response to a bewildering variety of stress conditions, such as heat shock, nutrient starvation, the presence of oxidants, DNA-damaging agents (including exposure to mitomycin C, cadmium, and hydrogen peroxide), as well as others, that may arrest cell growth. Proteins in the UspA family constitute a natural biological defense mechanism [1, 2]. Despite considerable research on the behavior of UspA family members, the biological and biochemical roles of these proteins remain largely uncharacterized. Very few details were available to help decipher their roles in the aforementioned cellular processes . A better understanding of the molecular mechanisms of E. coli’s UspA proteins is important for establishing effective therapeutic strategies. In particular, establishing the three-dimensional structural model of the UspE protein can provide hints to explore the function(s) of the UspA family.
E. coli has six small UspA superfamily genes: uspA, -C -D, -E, -F, and -G. To date, these proteins have been extensively investigated. Previous studies have shown that UspA family members show immaculate similarity. They encode either a small USP protein (approximately 14 to 15 kDa) that consists of two USP domains in tandem or a larger version (approximately 30 kDa) that consists of two peptides attached as a single functional protein [3, 4]. UspA, UspC, and UspD belong to class I; UspF and UspG belong to class II; two Usp domains of UspE belong to class II and IV based on the sequence and structural analysis [3, 5, 6]. Previous works have found that while Usp family members have partially overlapping functions, the functions of class I, II, and IV Usps are distinct . UspA proteins differ in their responses to protect cells from oxidative stress and DNA damage agents; UspA, UspC, UspD, and UspE are induced by exposure to mitomycin C, cadmium, and hydrogen peroxide. However, class II proteins, UspG and UspF, were associated with iron scavenging in the cell . As mentioned before, UspE is a tandem-type USP. When UspE proteins are split apart and treated separately, the UspE2 domain is more closely related to UspF and UspG. This is clearly visible in both the clustering analysis and the reconstructed cladogram. In contrast, UspE1 groups are more closely related to class I UspA proteins (UspACD) .
This paper includes structural and functional studies on UspE from E. coli. Specifically, it presents the three-dimensional X-ray crystal structure of the recombinantly produced UspE from E. coli at 3.2 Å resolution. Additionally, through the use of structural biochemical analyses, the UspE mechanisms were determined. In terms of its overall structure, UspE was found to be similar to the tandem-type Usp protein PMl1202 from Proteus mirabilis, which has a hydrophobic cavity that binds an unidentified ligand. It was also observed that UspE has an ATP binding motif (Gly269-Thr-Val-Gly272-X9-Gly282-Asn) in its C-terminal domain. The ATPase activity was then measured to determine if UspE had ATPase activity and to characterize UspE activity. Because previous research found that UspE is critical for Cd2+ defense, we characterized the role of UspE as part of the Cd2+ binding process by ITC and structural analysis and found that UspE has two Cd2+ binding sites in its tandem USP domain. These observations suggest that UspE performs several distinct functions, such as ATP hydrolysis and cadmium defense. Although the molecular function of this protein remains unknown, our three-dimensional structures of UspE offer valuable clues to understand its potential biochemical mechanisms.
Structure determination, refinement and protein data bank accession number of UspE
Beamline 5C, Pohang Accelerator Laboratory
a = b = 121.1 Å, c = 241.7 Å, α = β = γ = 90o
15.3 % (33.4 %)
Average I/σ (I)
Rmsd for bonds (Å)
Rmsd for angles (◦)
Ramachandran plot (%)
Number of atoms
Average B factor (Å2)
Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) measurements were performed on a Microcal iTC 200 (GE Healthcare) VP-ITC microcalorimeter at 298 K. The protein was dialyzed against 20 mM Hepes (pH 7.0) and 150 mM NaCl. The titration CdCl2 solution was prepared with 20 mM Hepes (pH 7.0) and 150 mM NaCl by adding 2 mM CdCl2. Both the protein and the titrant CdCl2 solutions were thoroughly degassed in a ThermoVac apparatus (Microcal). The titration reaction was performed by sequential injections of 40 μl CdCl2 solution into the sample cell. The duration of the injection was 120 s. The syringe was rotated at 600 rev min-1. Triplet measurements were collected in each case.
Kinase-Glo® luminescent kinase assay
The in vitro ATPase activity of UspE was measured by quantifying the amount of ATP remaining in the solution following a kinase reaction using a Kinase-Glo® Luminescent Kinase Assay Kit (Promega, Fitchburg, WI, USA). The assay was performed in a 96-well plate in a kinase reaction volume of 50 μl containing 10 mM MgCl2,5 μM ATP,10 mM HEPES (pH 8.0) and 150 mM NaCl. The reaction was initiated by adding the protein to a final concentration of 0.4 mg/ml-3.2 mg/ml. The reaction mixture was kept at 310 K for 20 min in a water bath. Reaction mixtures containing no UspE were used as negative controls. The kinase reaction mixture was incubated with 50 μl of ATP detection reagent. The plates were then incubated for another 10 min at 310 K. The Synergy2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) was used to collect the relative light unit (RLU) signal. The luminescent signal was positively correlated with the amount of remaining ATP and inversely correlated with the amount of kinase activity.
Results and discussion
E. coli UspE accommodates an unidentified ligand
E. coli UspE has an ATP binding motif and ATPase activity
UspE has putative Cd2+ binding sites
ITC experiment Cd2+ binding to UspE
In summary, the crystal structure of UspE from E. coli is representative of a tandem-type USP. The crystal structure of E. coli UspE reveals a hydrophobic pocket that moderately binds an unidentified ligand. Combined with previous studies, we can conclude that UspE is probably related to an intermediate in lipid A biosynthesis. We subsequently found through the sequence analysis that UspE has an ATP binding motif (Gly269- X2-Gly272-X9-Gly282-Asn) in the C-terminal domain and has ATPase activity, though this did not appear in the crystal structure. We were also able to perform an ITC experiment which revealed that UspE probably has two Cd2+ binding sites and that the His117, His119, His193, and His244 residues within the β-barrel domain are critical for binding Cd2+. We believe that this information is a significant contribution to understanding the molecular mechanisms of E. coli UspE.
In this study, we have determined the crystal structure of UspE of E. coli as a representative of a tandem-type USP. The UspE consists of two tandem USP domains that are highly conserved in this protein family. We found a hydrophobic pocket in the UspE structure, which was strongly bound to unidentified ligand. Combined with a previous study, evidence suggests that the UspE is related to an intermediate in lipid A biosynthesis. We subsequently found that sequence analysis suggests that UspE has an ATP binding motif (Gly269- X2-Gly272-X9-Gly282-Asn) in the C-terminal domain of UspE and has ATPase activity, but this was not confirmed by the crystal structure. We were also able to perform the ITC experiment, which revealed that the UspE probably has two Cd2+ binding sites, comprised of the His117, His 119, His193, and His244 residues within the β-barrel domain. Both of them are essential for Cd2+ binding to UspE protein. As discussed before, USPs might be associated with several functions, such as cadmium binding, ATPase activity, and an intermediate in lipid A biosynthesis.
- E. coli :
isothermal titration calorimetry
- P. mirabilis :
protein data bank
relative light unit
- S. enterica serovar Typhimurim:
Salmonella enterica serovar Typhimurium
universal stress protein
We gratefully acknowledge the access to the beamline 5C at Pohang Light Source (PLS) (Pohang, South Korea). This study was supported by the National Natural Science Foundation of China (Grant No. 31200556 to Y. Xu, Grant No.31301447 to Y. Ji, and Grant No. 21172028 to Shengdi Fan), the China Postdoctoral Science Foundation (Grant No. 2013 M540229 to Y. Xu), and the Fundamental Research Funds for the Central Universities (Grant No. DC201502020203 to Y. Xu).
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