Thermostability of multidomain proteins: elongation factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms.


Abstract

Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF-Tus, their isolated G-domains, and six chimeric EF-Tus composed of domains of either EF-Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized. BstEF-Tu and BstG-domain bound GDP and GTP with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of EcEF-Tu. In contrast, the EcG-domain bound the nucleotides with much lower, micromolar affinities. The exchange of domains 2 and 3 had essentially no effect on the GDP-binding activity; all complexes of chimeric EF-Tus with GDP retained K(d) values in the nanomolar range. The final thermostability level of either EF-Tu was the result of a cooperative interaction between the G-domains and domains 2 + 3. The G-domains set up a "basic" level of the thermostability, which was approximately 20 degrees C higher with the BstG-domain than with the EcG-domain. This correlated with the growth temperature optimum difference of both bacteria and two distinct thermostabilization features of the BstG-domain: an increase of charged residues at the expense of polar uncharged residues (CvP bias), and a decrease in the nonpolar solvent-accessible surface area. Domains 2 + 3 contributed by further stabilization of alpha-helical regions and, in turn, the functions of the G-domains to the level of the respective growth temperature optima. Their contributions were similar irrespective of their origin but, with Ecdomains 2 + 3, dependent on the guanine nucleotide binding state. It was lower in the GTP conformation, and the mechanism involved the destabilization of the alpha-helical regions of the G-domain by Ecdomain 2. Study holds ProTherm entries: 16803, 16804, 16805, 16806, 16807, 16808, 16809, 16810, 16811, 16812, 16813, 16814, 16815, 16816 Extra Details: Protein in GDP conformation. 10 mM 2-mercaptoethanol was added in the experiment. EF-Tu; thermostability; chimeric protein; EF-Tu domains; G-domain; Escherichia coli; Bacillus stearothermophilus

Submission Details

ID: zXResXRS3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Sanderová H;Hůlková M;Malon P;Kepková M;Jonák J,Protein Sci. (2004) Thermostability of multidomain proteins: elongation factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms. PMID:14691225
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4PC3 2015-05-06 1.83 Elongation factor Tu:Ts complex with partially bound GDP
1QZD 2003-11-04 10.0 EF-Tu.kirromycin coordinates fitted into the cryo-EM map of EF-Tu ternary complex (GDP.Kirromycin) bound 70S ribosome
3EQ4 2008-12-16 12.0 Model of tRNA(Leu)-EF-Tu in the ribosomal pre-accommodated state revealed by cryo-EM
4V6L 2014-07-09 13.2 Structural insights into cognate vs. near-cognate discrimination during decoding.
1LS2 2002-06-26 16.8 Fitting of EF-Tu and tRNA in the Low Resolution Cryo-EM Map of an EF-Tu Ternary Complex (GDP and Kirromycin) Bound to E. coli 70S Ribosome
5JBQ 2016-07-13 2.01 EF-TU (ESCHERICHIA COLI) IN COMPLEX WITH THIOMURACIN ANALOG
1EFC 1999-03-18 2.05 INTACT ELONGATION FACTOR FROM E.COLI
3U6B 2012-02-08 2.12 Ef-tu (escherichia coli) in complex with nvp-ldi028
2HCJ 2006-10-31 2.12 Trypsin-modified Elongation Factor Tu in complex with tetracycline
5MI8 2017-12-20 2.18 Structure of the phosphomimetic mutant of EF-Tu T383E
4G5G 2012-12-26 2.3 ef-tu (Escherichia coli) complexed with nvp-ldu796
2BVN 2005-09-01 2.3 E. coli EF-Tu:GDPNP in complex with the antibiotic enacyloxin IIa
1D8T 2000-10-25 2.35 CRYSTAL STRUCTURE OF ELONGATION FACTOR, TU (EF-TU-MGGDP) COMPLEXED WITH GE2270A, A THIAZOLYL PEPTIDE ANTIBIOTIC
5I4Q 2017-06-28 2.35 Contact-dependent inhibition system from Escherichia coli NC101 - ternary CdiA/CdiI/EF-Tu complex (domains 2 and 3)
3AVX 2012-01-18 2.41 Structure of viral RNA polymerase complex 5
3U6K 2012-02-08 2.45 Ef-tu (escherichia coli) in complex with nvp-ldk733
6EZE 2018-08-22 2.47 The open conformation of E.coli Elongation Factor Tu in complex with GDPNP.
1DG1 1999-12-01 2.5 WHOLE, UNMODIFIED, EF-TU(ELONGATION FACTOR TU).
3MMP 2010-06-09 2.5 Structure of the Qb replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins
1EFU 1997-01-11 2.5 ELONGATION FACTOR COMPLEX EF-TU/EF-TS FROM ESCHERICHIA COLI
3VNV 2012-08-08 2.6 Complex structure of viral RNA polymerase II
3AVW 2012-01-18 2.6 Structure of viral RNA polymerase complex 4
3AVT 2012-01-18 2.61 Structure of viral RNA polymerase complex 1
3AVY 2012-01-18 2.62 Structure of viral RNA polymerase complex 6
1EFM 1987-07-16 2.7 STRUCTURE OF THE GDP DOMAIN OF EF-TU AND LOCATION OF THE AMINO ACIDS HOMOLOGOUS TO RAS ONCOGENE PROTEINS
3U2Q 2012-05-02 2.7 EF-Tu (Escherichia coli) in complex with NVP-LFF571
5OPD 2018-08-29 2.75 Structure of phosphorylated EF-Tu in complex with GTP
3AGP 2010-09-01 2.8 Structure of viral polymerase form I
2HDN 2006-10-31 2.8 Trypsin-modified Elongation Factor Tu in complex with tetracycline at 2.8 Angstrom resolution
5MI3 2017-12-20 2.8 Structure of phosphorylated translation elongation factor EF-Tu from E. coli
5AFI 2015-03-11 2.9 2.9A Structure of E. coli ribosome-EF-TU complex by cs-corrected cryo-EM
4Q7J 2014-08-27 2.9 Complex structure of viral RNA polymerase
1ETU 1988-07-16 2.9 STRUCTURAL DETAILS OF THE BINDING OF GUANOSINE DIPHOSPHATE TO ELONGATION FACTOR TU FROM E. COLI AS STUDIED BY X-RAY CRYSTALLOGRAPHY
3AVU 2012-01-18 2.91 Structure of viral RNA polymerase complex 2
5WFS 2018-05-02 3.0 70S ribosome-EF-Tu H84A complex with GTP and near-cognate tRNA (Complex C4)
5WDT 2018-04-25 3.0 70S ribosome-EF-Tu H84A complex with GppNHp
5WE4 2018-04-25 3.1 70S ribosome-EF-Tu wt complex with GppNHp
3AVV 2012-01-18 3.12 Structure of viral RNA polymerase complex 3
4FWT 2012-08-08 3.2 Complex structure of viral RNA polymerase form III
5UYM 2017-06-07 3.2 70S ribosome bound with cognate ternary complex base-paired to A site codon, closed 30S (Structure III)
3VNU 2012-08-08 3.2 Complex structure of viral RNA polymerase I
4R71 2015-09-23 3.21 Structure of the Qbeta holoenzyme complex in the P1211 crystal form
3AGQ 2010-09-01 3.22 Structure of viral polymerase form II
5MI9 2017-12-20 3.3 Structure of the phosphomimetic mutant of the elongation factor EF-Tu T62E
5I4R 2017-06-28 3.3 Contact-dependent inhibition system from Escherichia coli NC101 - ternary CdiA/CdiI/EF-Tu complex (trypsin-modified)
1OB2 2004-05-27 3.35 E. coli elongation factor EF-Tu complexed with the antibiotic kirromycin, a GTP analog, and Phe-tRNA
5WFK 2018-05-02 3.4 70S ribosome-EF-Tu H84A complex with GTP and near-cognate tRNA (Complex C3)
5WE6 2018-04-25 3.4 70S ribosome-EF-Tu H84A complex with GTP and cognate tRNA
2FX3 2006-03-28 3.4 Crystal Structure Determination of E. coli Elongation Factor, Tu using a Twinned Data Set
5WF0 2018-05-02 3.6 70S ribosome-EF-Tu H84A complex with GTP and near-cognate tRNA (Complex C2)
5UYL 2017-06-14 3.6 70S ribosome bound with cognate ternary complex base-paired to A site codon (Structure II)
4PC7 2015-05-06 3.6 Elongation factor Tu:Ts complex in a near GTP conformation.
5UYQ 2017-06-07 3.8 70S ribosome bound with near-cognate ternary complex base-paired to A site codon, closed 30S (Structure III-nc)
5UYP 2017-06-07 3.9 70S ribosome bound with near-cognate ternary complex base-paired to A site codon, open 30S (Structure II-nc)
5UYK 2017-06-07 3.9 70S ribosome bound with cognate ternary complex not base-paired to A site codon (Structure I)
5UYN 2017-06-07 4.0 70S ribosome bound with near-cognate ternary complex not base-paired to A site codon (Structure I-nc)
4V69 2014-07-09 6.7 Ternary complex-bound E.coli 70S ribosome.
4V6K 2014-07-09 8.25 Structural insights into cognate vs. near-cognate discrimination during decoding.
3EQ3 2008-12-16 9.0 Model of tRNA(Trp)-EF-Tu in the ribosomal pre-accommodated state revealed by cryo-EM
3EP2 2008-12-16 9.0 Model of Phe-tRNA(Phe) in the ribosomal pre-accommodated state revealed by cryo-EM

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.4 Elongation factor Tu 1 O31301 EFTU_BUCSC
90.1 Elongation factor Tu 1 C4K4F8 EFTU_HAMD5
90.1 Elongation factor Tu 1 B8D851 EFTU_BUCAT
90.1 Elongation factor Tu 1 O31297 EFTU_BUCAI
90.1 Elongation factor Tu 1 B8D9U9 EFTU_BUCA5
92.1 Elongation factor Tu 1 P57966 EFTU2_PASMU
91.1 Elongation factor Tu 1 A0KQ95 EFTU_AERHH
92.4 Elongation factor Tu 1 B0UV21 EFTU_HISS2
92.4 Elongation factor Tu 1 Q0I1U9 EFTU_HAES1
91.1 Elongation factor Tu 1 O31298 EFTU_BUCAP
93.1 Elongation factor Tu 1 B0BQZ3 EFTU_ACTPJ
93.1 Elongation factor Tu 1 A3N246 EFTU_ACTP2
92.1 Elongation factor Tu 1 A6VKH7 EFTU_ACTSZ
92.9 Elongation factor Tu 1 P57939 EFTU1_PASMU
93.4 Elongation factor Tu 1 Q7TTF9 EFTU_HAEDU
92.9 Elongation factor Tu 1 P43926 EFTU_HAEIN
92.9 Elongation factor Tu 1 A5UHC1 EFTU_HAEIG
92.9 Elongation factor Tu 1 A5U9R1 EFTU_HAEIE
92.9 Elongation factor Tu 1 Q4QMT5 EFTU2_HAEI8
92.6 Elongation factor Tu 1 Q65QG6 EFTU_MANSM
93.1 Elongation factor Tu 1 Q4QMW6 EFTU1_HAEI8
93.9 Elongation factor Tu 1 Q7N9B1 EFTU1_PHOLL
93.4 Elongation factor Tu 1 Q1CN86 EFTU1_YERPN
93.6 Elongation factor Tu 1 Q2NQL7 EFTU_SODGM
93.6 Elongation factor Tu 1 A4TS36 EFTU2_YERPP
93.6 Elongation factor Tu 1 Q8ZAN8 EFTU2_YERPE
93.6 Elongation factor Tu 1 Q1C1T4 EFTU2_YERPA
93.6 Elongation factor Tu 1 Q66FQ9 EFTU1_YERPS
93.6 Elongation factor Tu 1 A7FNJ0 EFTU1_YERP3
94.4 Elongation factor Tu 1 Q7MYE8 EFTU2_PHOLL
94.1 Elongation factor Tu 1 A1JS52 EFTU2_YERE8
94.7 Elongation factor Tu 1 A1JIH3 EFTU1_YERE8
94.7 Elongation factor Tu 1 A8GKK1 EFTU2_SERP5
94.9 Elongation factor Tu 1 A8G8E0 EFTU1_SERP5
95.4 Elongation factor Tu 1 Q664R7 EFTU2_YERPS
95.4 Elongation factor Tu 1 Q1CCT9 EFTU2_YERPN
95.4 Elongation factor Tu 1 A7FNN8 EFTU2_YERP3
95.4 Elongation factor Tu 1 A4TGY7 EFTU1_YERPP
95.4 Elongation factor Tu 1 Q8ZJB2 EFTU1_YERPE
95.4 Elongation factor Tu 1 Q1C2U1 EFTU1_YERPA
95.7 Elongation factor Tu 1 Q6CZW6 EFTU_PECAS
96.9 Elongation factor Tu 1 A4W5A0 EFTU_ENT38
98.2 Elongation factor Tu 1 A6TEX7 EFTU_KLEP7
98.5 Elongation factor Tu 1 A7MKI5 EFTU_CROS8
99.7 Elongation factor Tu 1 A7ZUJ2 EFTU2_ECO24
100.0 Elongation factor Tu 1 P0A6N2 EFTU_ECOL6
100.0 Elongation factor Tu 1 P0A6N3 EFTU_ECO57
100.0 Elongation factor Tu 1 Q3YV04 EFTU2_SHISS
100.0 Elongation factor Tu 1 Q0SY20 EFTU2_SHIF8
100.0 Elongation factor Tu 1 Q1R5U4 EFTU2_ECOUT
100.0 Elongation factor Tu 1 P0CE48 EFTU2_ECOLI
100.0 Elongation factor Tu 1 B1IVA7 EFTU2_ECOLC
100.0 Elongation factor Tu 1 Q0TA85 EFTU2_ECOL5
100.0 Elongation factor Tu 1 A1AIF3 EFTU2_ECOK1
100.0 Elongation factor Tu 1 A8A779 EFTU2_ECOHS
99.7 Elongation factor Tu 1 Q0SZX8 EFTU1_SHIF8
99.7 Elongation factor Tu 1 P0A1H5 EFTU_SALTY
99.7 Elongation factor Tu 1 P0A1H6 EFTU_SALTI
99.7 Elongation factor Tu 1 A9MT05 EFTU_SALPB
99.7 Elongation factor Tu 1 Q5PIW4 EFTU_SALPA
99.7 Elongation factor Tu 1 Q57H76 EFTU_SALCH
99.7 Elongation factor Tu 1 A9MHG0 EFTU_SALAR
100.0 Elongation factor Tu 1 Q83JC4 EFTU_SHIFL
100.0 Elongation factor Tu 1 Q32B27 EFTU_SHIDS
100.0 Elongation factor Tu 1 Q31VV0 EFTU_SHIBS
100.0 Elongation factor Tu 1 Q3YWT3 EFTU1_SHISS
100.0 Elongation factor Tu 1 Q1R5Y2 EFTU1_ECOUT
100.0 Elongation factor Tu 1 P0CE47 EFTU1_ECOLI
100.0 Elongation factor Tu 1 B1IPW0 EFTU1_ECOLC
100.0 Elongation factor Tu 1 Q0TCC0 EFTU1_ECOL5
100.0 Elongation factor Tu 1 A1AGM6 EFTU1_ECOK1
100.0 Elongation factor Tu 1 A8A5E6 EFTU1_ECOHS
100.0 Elongation factor Tu 1 A7ZSL4 EFTU1_ECO24
96.7 Elongation factor Tu B7GJ65 EFTU_ANOFW
96.5 Elongation factor Tu C5D3R5 EFTU_GEOSW
98.2 Elongation factor Tu A4IJI7 EFTU_GEOTN
99.2 Elongation factor Tu Q5L3Z9 EFTU_GEOKA
100.0 Elongation factor Tu O50306 EFTU_GEOSE