NMR STRUCTURE NOTE

The C-terminal region of human eukaryotic elongation factor 1Bd

Huiwen Wu1 • Chen Wang2,3,4,5 • Weibin Gong1 • Jinfeng Wang1 • Jinsong Xuan5 •

Sarah Perrett1 • Yingang Feng2,3,4

Received: 29 October 2015 / Accepted: 6 January 2016 / Published online: 13 January 2016

� Springer Science+Business Media Dordrecht 2016

Biological context

In the elongation step of eukaryotic protein biosynthesis,

the eukaryotic elongation factor 1A (eEF1A) in its GTP-

bound active state transports the aminoacyl tRNA (aa-

tRNA) to the A site of the ribosome (Sasikumar et al.

2012). Correct codon–anticodon pairing induces hydrolysis

of GTP to GDP, which results in a conformational change

of eEF1A that causes its release from both the ribosome

and aa-tRNA. The eukaryotic elongation factor 1B

(eEF1B) complex containing 2–4 subunits helps to enhance

the intrinsically slow (*0.7 9 10-3/s) dissociation rate of

GDP from eEF1A by approximately 3000-fold and results

in GTP reloading and eEF1A reactivation (Janssen and

Moller 1988). The eEF1B complex is comprised of one or

two guanine nucleotide exchange factors (GEFs) (EF1Baexists in all eukaryotes, eEF1Bd exists only in metazoans,

and eEF1Bb exists only in plants), a scaffold component

named eEF1Bc, and a valine-tRNA synthetase (Val-RS)

additionally in metazoans (Le Sourd et al. 2006). Because

there is no structure of the eEF1B complex available,

several models have been proposed to explain the assembly

of the eEF1B complex in different species (Janssen et al.

1994; Sheu and Traugh 1997; Mansilla et al. 2002).

The GEFs of the eEF1B complex are the catalytic

components and each of them contain two regions (van

Damme et al. 1990; Wu et al. 2015): a less conserved

N-terminal region (Sanders et al. 1993) and a highly con-

served C-terminal region which comprises a central acidic

region, termed the CAR domain, and a C-terminal catalytic

GEF domain. The N-terminal region is mainly responsible

for interacting with eEF1Bc to facilitate assembly of the

eEF1B complex. In the C-terminal region, the GEF domain

is an essential catalytic domain, while the CAR domain is

not essential for the exchange activity. However, it was

found that the CAR domain may enhance the exchange

activity and regulates the GEF activity (Perez et al. 1998;

van Damme et al. 1991). The CAR domain contains the

casein kinase 2 (CK2) phosphorylation site (Sheu and

Traugh 1997) and has been shown to interact with trans-

lationally-controlled tumor protein (TCTP) which inhibits

the nucleotide-exchange activity of eEF1Bd (Wu et al.

2015; Cans et al. 2003). Even though the GEF and CAR

domains are highly conserved in eEF1Ba, eEF1Bb, and

eEF1Bd, evidence suggests that the regulation of their

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10858-016-0012-6) contains supplementarymaterial, which is available to authorized users.

& Sarah Perrett

sarah.perrett@cantab.net

& Yingang Feng

fengyg@qibebt.ac.cn

1 National Laboratory of Biomacromolecules, Institute of

Biophysics, Chinese Academy of Sciences, Beijing 100101,

China

2 Qingdao Engineering Laboratory of Single Cell Oil, Qingdao

Institute of Bioenergy and Bioprocess Technology, Chinese

Academy of Sciences, 189 Songling Road,

Qingdao 266101, Shandong, China

3 Shandong Key Laboratory of Synthetic Biology, Qingdao

Institute of Bioenergy and Bioprocess Technology, Chinese

Academy of Sciences, 189 Songling Road,

Qingdao 266101, Shandong, China

4 CAS Key Laboratory of Biofuels, Qingdao Institute of

Bioenergy and Bioprocess Technology, Chinese Academy of

Sciences, 189 Songling Road, Qingdao 266101, Shandong,

China

5 Department of Biological Science and Engineering, School of

Chemical and Biological Engineering, University of Science

and Technology Beijing, Beijing 100083, China

123

J Biomol NMR (2016) 64:181–187

DOI 10.1007/s10858-016-0012-6

activity and the mechanism of binding to eEF1A could be

different. For example, human eEF1Ba but not eEF1Bdcan complement eEF1Ba-deficient yeasts (Carr-Schmid

et al. 1999); binding of eEF1A to eEF1Ba but not eEF1Bdresults in masking of the CK2 phosphorylation site (Sheu

and Traugh 1997); and the GEF activity of eEF1Ba but not

eEF1Bd increases after binding eEF1Bc (Bec et al. 1994).

The structures of the catalytic GEF domain of human

eEF1Ba (eEF1Ba GEF) and the yeast eEF1A-eEF1Ba GEF

domain complex have been reported (Perez et al. 1999;

Andersen et al. 2000, 2001). According to the reported

structures, eEF1Ba GEF is a two layered a/b sandwich

containing a b-sheet with four antiparallel strands and two

helices packing against one face of the b-sheet. The structure

of the yeast eEF1A-eEF1Ba GEF domain complex reveals

that eEF1A contains three domains (1, 2 and 3). One edge of

eEF1Ba GEF, which consists of N- and C- termini, interacts

with domain 1 of eEF1A, while the opposite edge of eEF1BaGEF interacts with domain 2 of eEF1A. The three residues at

the C-terminal end of eEF1Ba, especially the absolutely

conserved lysine residue, are essential for releasing GDP

from eEF1A, as it forms contacts with the domain 1 of

eEF1A at a Mg2? binding position, the displacement of

which triggers the release of nucleotide. However, a recent

crystallography study of human eEF1A2 suggests that the

exchange mechanism for human eEF1A may be different

and the Mg2? removal is dispensable for GDP binding and

dissociation (Crepin et al. 2014). The structure of the CAR

domain of human eEF1Bd was determined recently (Wu

et al. 2015). The CAR domain, containing an a-helix and

two flexible loops, is structurally independent of the GEF

domain as suggested by the interaction study. However,

neither the structure of the whole conserved C-terminal

region of eEF1B GEFs nor the structure of the GEF domain

of eEF1Bd or eEF1Bb is currently available.

Here, we report the NMR structures of the CAR and

GEF domains in the C-terminal region of human eEF1Bd.

Combined with the previously determined GEF structures,

the GEF domain structure of human eEF1Bd obtained here

was used for detailed structural comparisons of GEFs in

different species, in order to investigate the functions of

different domains of eEF1B GEFs.

Methods and results

Protein expression and purification

The gene construction, protein expression and purification

of human eEF1Bd CAR-GEF (residues 153–281 of

eEF1Bd) were performed following the procedures previ-

ously described (Wu et al. 2015). Briefly, the gene fragment

Table 1 Restraints and structure statistics of the 20 lowest energy

conformers of human eEF1Bd CAR-GEF

NOE restraints

Intra-residue 694

Sequential 424

Medium-range 234

Long-range 518

Ambiguous 1118

Total 2988

Hydrogen bond restraints 106

Torsion angle restraints

Phi (u) 101

Psi (w) 101

Chi1 (v1) 47

Violations

Max. NOE violation (A) 0.200

Max. torsion angle violation (�) 3.79

R.M.S.D. from mean structure (A)a

All residues of the GEF domain (residues 193–281)

Backbone heavy atoms 0.45 ± 0.10

All Heavy atoms 0.82 ± 0.06

Regular secondary structure residues of the GEF domainb

Backbone heavy atoms 0.31 ± 0.05

All heavy atoms 0.69 ± 0.05

All residues of the CAR domain (residues 153–192)

Backbone heavy atoms 7.12 ± 1.27

All heavy atoms 7.69 ± 1.23

Regular secondary structure residues of the CAR domainc

Backbone heavy atoms 0.49 ± 0.20

All heavy atoms 1.46 ± 0.17

Ramachandran statistics

Most favored region (%) 86.5

Additionally allowed (%) 11.1

Generously allowed (%) 1.0

Disallowed (%) 1.3d

WHAT_CHECK Z-scores

1st generation packing quality -0.823

2nd generation packing quality -1.546

Ramachandran plot appearance -2.630

Chi-1/chi-2 rotamer normality -1.838

Backbone conformation -1.346

Inside/outside distribution 1.128

a The structures were superposed by the backbone heavy atoms of

regular secondary structure regions of the corresponding domain

before calculating R.M.S.D of the GEF or CAR domainb Regular secondary structure regions of the GEF domain are resi-

dues 195–204, 211–219, 226–235, 241–250, 256–266, 270–280c Regular secondary structure regions of the CAR domain are resi-

dues 169–185d All the residues in disallowed regions of the Ramachandran plot are

located in the disordered N-terminal loop and the linker between the

CAR and GEF domains

182 J Biomol NMR (2016) 64:181–187

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of human eEF1Bd CAR-GEF was cloned into a modified

pET28a expression vector and the protein was expressed in

Escherichia coli BL21 (DE3). Proteins were first purified by

affinity chromatography using a Ni2?-column (Chelating

Sepharose Fast Flow, GE Healthcare), and then were

digested with PreScission protease to remove the N-terminal

tag by passing through the Ni2?-column again. The fraction

containing eEF1Bd CAR-GEF was further purified using a

Superdex 75 gel filtration column (GE Healthcare).

NMR spectroscopy

Uniformly 15N- or 15N/13C-labeled human eEF1Bd CAR-

GEF was used in preparation of the NMR sample for dif-

ferent experiments. Human eEF1Bd CAR-GEF protein

(0.8 mM) was dissolved in 10 % (v/v) D2O containing

20 mM Tris–HCl buffer (pH 7.5), 200 mM NaCl, and

0.01 % 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). All

NMR experiments were performed at 298 K on a Bruker

DMX 600 MHz NMR spectrometer equipped with a cryo-

probe. Two-dimensional 1H-15N and 1H-13C HSQC, and

three-dimensional CBCA(CO)NH, HNCACB, HNCO,

HN(CA)CO, HBHA(CO)NH, HBHANH, HCCH-TOCSY,

CCH-COSY, and CCH-TOCSY spectra were acquired for

backbone and side-chain resonance assignments. Distance

restraints were derived from three-dimensional 1H-15N and1H-13C NOESY-HSQC spectra collected with mixing times

of 150 ms. All data were processed with FELIX (Accelrys

Inc.) or NMRPipe (Delaglio et al. 1995) and analyzed with

NMRViewJ (Johnson and Blevins 1994).

Fig. 1 NMR structures of the

C-terminal region containing

the CAR and GEF domains of

human eEF1Bd (human

eEF1Bd CAR-GEF).

a Superposition of 20 lowest-

energy conformers for best

fitting to the backbone of the

CAR domain. b The same

superposition as in (a) for best

fitting to the backbone of the

GEF domain. c Ribbon

presentation of the structure of

human eEF1Bd CAR-GEF. The

secondary structure elements

are labeled. d Electrostatic

potential surface of the CAR

domain. The dashed curves

indicate the surfaces of

disordered regions which should

not be considered as defined

surfaces. e Electrostatic

potential surface of the GEF

domain. The CAR domain is

shown in blue and the GEF

domain in red (a, b, and c). In

the electrostatic surfaces, red is

negatively charged, and blue is

positively charged

J Biomol NMR (2016) 64:181–187 183

123

Structure calculations

The structures of human eEF1Bd CAR-GEF were initially

calculated with the program CYANA (Herrmann et al.

2002), and then refined using CNS (Brunger et al. 1998)

with semi-automated NOE assignments by SANE (Duggan

et al. 2001). Backbone u and w and side chain v1 dihedral

angle restraints obtained using TALOS-N (Shen and Bax

2013) and hydrogen-bond restraints according to the reg-

ular secondary structure patterns were also incorporated

into the late-stage structural calculation. The 50 lowest

energy conformers were selected from 100 initial structures

for refinement in explicit water using RECOORDScript

(Nederveen et al. 2005), and then the 20 lowest energy

structures were chosen to represent the final ensemble. The

quality of these structures was analyzed using the programs

MOLMOL (Koradi et al. 1996), PROCHECK-NMR

(Laskowski et al. 1996), and WHATCHECK (Hooft et al.

1996). PyMOL (Schrodinger, LCC) was used for visual-

ization of the structures. Atomic coordinates of the struc-

tures have been deposited in the PDB with accession code

2N51, and chemical shift assignments have been deposited

in the BioMagResBank under accession number 25690.

Solution structure of the C-terminal region

of human eEF1Bd

About 96.7 % of the 1H, 15N, and 13C resonances of the

backbone and side-chain atoms for human eEF1Bd CAR-

GEF were assigned. The assigned 1H-15N HSQC spectrum

is shown in Fig. S1. Based on the resonance assignments,

nearly 3000 NOEs were identified to generate distance

restraints for the structure calculations. However, no long

range NOEs between the CAR and GEF domains were

observed. The high quality of the final structural ensemble

of human eEF1Bd CAR-GEF was verified by statistical

analysis (Table 1). In Fig. 1a, b, the ensemble of structures

for each of the CAR and GEF domains are well superposed

when each domain is considered independently, and the

linking peptide segment between the two domains is dis-

ordered, showing that the two domains do not adopt a

unique orientation relative to each other. This suggests that

the CAR and GEF domains are structurally independent of

each other as reported previously (Wu et al. 2015). This is

further confirmed by the finding that the CAR and GEF

domains have different R2/R1 ratios in 15N backbone

relaxation measurements (Fig. S2).

The CAR domain (residues 153–192) contains one a-

helix (aCAR, residues 169–185) and two flexible loops on

either side of the helix (Fig. 1c). Although the NOE pat-

terns in the helix cannot be unambiguously verified

because of the severe overlap of the chemical shifts for

both HN and HA atoms in the helix, the helix is identified

by both chemical shift index (CSI) and TALOS-N pre-

diction from the chemical shifts (Fig. S3). The C-terminal

flexible loop represents a disordered region linking the

CAR and GEF domains. The electrostatic surface of the

CAR domain shows that the N-terminal flexible loop is

largely negatively charged while the C-terminal part of the

helix and the C-terminal flexible loop are mainly positively

charged (Fig. 1d). These structural features of the CAR

domain are essentially identical to those of the structure of

the standalone CAR domain reported in the previous study

(Wu et al. 2015), and the RMSD of backbone atoms in the

helices (residues 169–185) between the CAR domains of

the two structures is 0.73 ± 0.26 A. The GEF domain

(residues 193–281) contains an antiparallel four-strand b-

sheet (b1, 195–204; b2, 226–235; b3, 241–250; b4,

270–280) and two a-helices (a1, 211–219; a2, 256–266) in

the order b1–a1–b2–b3–a2–b4, which forms a typical two-

layer a/b sandwich structure (Fig. 1c). One side of the GEF

domain, including mainly the a2 and b4, is largely nega-

tively charged, while the other side is neutral and positively

charged (Fig. 1e).

Discussion and conclusions

The overall fold of the human eEF1Bd GEF domain is the

same as the previously reported structures of human and

yeast eEF1Ba GEF domains, which is expected as they

share high sequence homology (Fig. 2a). The human

eEF1Bd GEF domain (described here) and the eEF1BaGEF domain (PDB code 1B64) superimpose with an

RMSD of 1.6 A, indicating significant similarity between

the domains. Differences occur mainly in the flexible loops

and the boundary of secondary structural elements

(Fig. 2b), which is likely due to the fluctuation of solution

NMR structures. Although the domains show conserved

structural features, they do not share all conserved salt

bridges. Each domain has a salt bridge, which is between

residues E262 and K265 within the helix a2 of the eEF1BdGEF domain and residues K157 and E160 within the helix

a1 of the eEF1Ba GEF domain. However, the corre-

sponding residues are Q206 and A209 in the eEF1Ba GEF

domain, and Q213 and A216 in the eEF1Bd GEF domain.

The salt bridge formed by the non-conserved residues in

helix a2 of the domains may induce about one additional

turn in the helix a2 of the eEF1Bd GEF domain compared

to the eEF1Ba GEF domain (Fig. 2c). Nevertheless, no

significant backbone difference of the a1 helices is

observed (Fig. 2c). Since the GEF domain represents a

two-layer a/b sandwich structure (Fig. 1c), and the b-sheet

is responsible for binding to eEF1A, it is not clear whether

the non-conservation of the salt bridges in the helices has

any functional significance.

184 J Biomol NMR (2016) 64:181–187

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Superposition of the structures of the human eEF1BdGEF domain with the previously determined yeast eEF1BaGEF domain in the eEF1Ba-eEF1A complex (PDB code

1IJF) gives an RMSD of 1.3 A, revealing a highly

conserved overall fold (Fig. 2d, e). Residues involved in

catalysis and contact with eEF1A of the yeast eEF1BaGEF in the complex are highly conserved in the human

eEF1Bd and eEF1Ba GEF domains in term of sequence

Fig. 2 Structure comparison of human eEF1Bd CAR-GEF with

known eEF1B structures. a Sequence alignment of the CAR-GEFs of

human eEF1Bd, human eEF1Ba, and yeast eEF1Ba. Secondary

structure elements of human eEF1Bd CAR-GEF are shown above the

sequence. The residues in yeast eEF1Ba that contact eEF1A in the

eEF1A-eEF1Ba complex are indicated by stars below the sequence.

b Superposition of a representative set of the NMR structures of

human eEF1Bd GEF domain (red) onto human eEF1Ba GEF domain

(PDB code 1B64, cyan). c The salt bridges formed within the helices

of human eEF1Bd GEF domain (red) and human eEF1Ba GEF

domain (PDB code 1B64, cyan). Residues are shown as sticks colored

by atom types (carbon in green, oxygen in red, and nitrogen in blue).

d Superposition of the NMR structure of human eEF1Bd GEF domain

(red) onto the crystal structure of yeast eEF1Ba GEF domain (PDB

code 1IJF, green). The residues in yeast eEF1Ba that contact eEF1A

and the corresponding residues in human eEF1Bd are shown as sticks.

e Superposition of the NMR structure of human eEF1Bd CAR-GEF

onto the crystal structure of the yeast eEF1Ba-eEF1A complex (PDB

code 1IJF). The human eEF1Bd CAR domain, the human eEF1BdGEF domain, the yeast eEF1Ba GEF domain, and the yeast eEF1A

are colored in blue, red, green, and grey, respectively. GDP is shown

as orange sticks, and Mg2? is shown as a magenta ball

J Biomol NMR (2016) 64:181–187 185

123

and structure. These residues are located on two edges of

the antiparallel four-strand b-sheet (Fig. 2d). On one edge,

the contacting residues include the absolutely conserved

residues K128, P160, I165, Q196, and D199 at the C-ter-

mini of b1 and b2 and the N-termini of b3 and b4, as well as

the similar hydrophobic residues F163 and Y238 in the

human and yeast GEF domains, respectively, in the loop

between b1 and b2 (Fig. 2d). On the opposite edge of the b-

sheet, a group of conserved residues at the C-terminus of

b4, including the absolutely conserved residue K205 (K280

in yeast eEF1Ba), similar hydrophobic residues M203 and

L206 (F276 and I281 in yeast eEF1Ba), and similar glu-

tamine residue Q204 (N279 in yeast eEF1Ba) (Fig. 2d), are

involved in the interaction with eEF1A. This similarity

suggests that the modes of binding between different

eEF1B GEFs and eEF1A are essentially the same in

humans and yeast, and the different eEF1B GEFs may

share the same catalytic mechanism for GEF activity, that

is the C-terminal lysine residue of the eEF1B GEF domain

disrupts the interaction of Mg2? with eEF1A, resulting in

the release of GDP from eEF1A (Andersen et al. 2000,

2001). However, a recent study describing the crystal

structure of human eEF1A2 proposed a different exchange

mechanism for human eEF1A, namely that the conforma-

tional change induced by eEF1B binding plays a critical

role, rather than the Mg2? removal step (Crepin et al.

2014). As shown in the present structure comparison

(Fig. 2d), residues adjacent to the C-terminal lysine residue

in human eEF1Ba and eEF1Bd are similar but not identical

to those in yeast eEF1Ba. Presumably, the position and

conformation of the catalytic lysine of human eEF1Ba and

eEF1Bd may be slightly different from that of yeast

eEF1Ba when they bind to eEF1A, which might make the

Mg2? dispensable for GDP binding and dissociation.

In the antiparallel four-strand b-sheet of the GEF

domain, the C-terminal catalytic lysine residue is adjacent

to the N-terminus of the b1 strand of the GEF domain

(Fig. 2d). Since the CAR domain connects to the N-ter-

minus of the b1 strand of the GEF domain (Fig. 1c), such

an arrangement should form a basis for the regulatory role

of the CAR domain for the GEF activity reported previ-

ously (van Damme et al. 1991; Perez et al. 1998; Cans et al.

2003; Wu et al. 2015). Superposition of the human eEF1BdCAR-GEF structure onto the crystal structure of the yeast

eEF1Ba-eEF1A complex (Andersen et al. 2001) indicates

that the position of the CAR domain will be restricted to

some extent in the vicinity of the nucleotide binding pocket

of eEF1A when eEF1Bd and eEF1A form a complex

(Fig. 2e), which is probably the structural basis of the

regulation function of some CAR-domain binding proteins

such as TCTP (Wu et al. 2015). However, this cannot

exclude the possibility that the CAR domain also interacts

with eEF1A in the eEF1Bd-eEF1A complex to play a

regulatory role, because the linker between the CAR

domain and the GEF domain is flexible. If this is true, not

only the steric hindrance but also the disruption of the

interaction between the CAR domain and eEF1A could be

the mechanism of the guanine nucleotide dissociation

inhibitor activity of TCTP. Although clarification of the

functional role of the CAR domain needs further study, the

structure of human eEF1Bd CAR-GEF described here

provides a structural basis for understanding the guanine

nucleotide exchange function of eEF1Bd.

Acknowledgments This work was supported by the National Basic

Research Program from Ministry of Science and Technology of China

(973 Program, Grant Nos. 2012CB911000 and 2013CB910700 to

S.P.), the National High-tech R&D Program from Ministry of Science

and Technology of China (863 Program, Grant No. 2012AA02A707

to Y.F.), and the National Natural Science Foundation of China

(30800179 and 31170701 to Y.F.; 31300635 to J.X.; 31200578 and

31470747 to W.G.; and 31110103914 to S.P.).

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

Ethical standard Research does not involve human participants

and/or animals.

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