Crystal structure of the nucleotide-bindingdomain of mortalin, the mitochondrialHsp70 chaperone

Joseph Amick,1 Simon E. Schlanger,1 Christine Wachnowsky,2 Mitchell A.Moseng,3 Corey C. Emerson,3 Michelle Dare,1 Wen-I Luo,2 Sujay S. Ithychanda,1

Jay C. Nix,4 J. A. Cowan,2 Richard C. Page,3* and Saurav Misra1*

1Department of Molecular Cardiology, The Cleveland Clinic, Cleveland, Ohio 441952Department of Chemistry, The Ohio State University, Columbus, Ohio 432103Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 450564Molecular Biology Consortium, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720

Received 9 March 2014; Accepted 18 March 2014

DOI: 10.1002/pro.2466Published online 29 March 2014 proteinscience.org

Abstract: Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of

processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis,mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell

stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077.

Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and asubstrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 A

resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70

family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson’s disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation.

We present structure-based models for how the Mortalin-NBD may interact with the nucleotide

exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understandingof disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.

Keywords: mitochondria; protein quality control; Heat-shock protein 70; p53; chaperone inhibitor;

nucleotide binding

Introduction

The Hsp70 chaperone family plays key roles in cellu-

lar homeostasis and stress response. Hsp70-family

members not only regulate folding of nascent pro-

teins but also prevent protein aggregation, promote

disaggregation, and refold misfolded proteins. Hsp70-

family members participate in additional cellular

processes including conformational control of regula-

tory proteins, intracellular trafficking, uncoating of

clathrin-coated vesicles, and protein translocation

across intracellular membranes.1,2 In eukaryotes,

family members localize to the cytosol (Hsp70, Hsc70

and close homologues), the endoplasmic reticulum

(BiP/Grp78), and mitochondria (Mortalin).

Mortalin, also known as mtHsp70, HSPA9/

HSPA9B or Grp75, was identified in mice as a cellu-

lar mortality factor3 and determined to be an

Hsp70-family member primarily localized to mito-

chondria.4 Its sequence is slightly more

Additional Supporting Information may be found in the onlineversion of this article.

Grant sponsor: U.S. National Institutes of Health (S.M.); Grantnumber: R01-GM080271; Grant sponsor: National Institutes ofHealth (R.C.P.); Grant number: T32-HL007914. Grant sponsor:U.S. Department of Energy, Office of Basic Energy Sciences(The Advanced Light Source); Grant number: DE-AC03-76SF00098.

*Correspondence to: Saurav Misra; Department of MolecularCardiology, The Cleveland Clinic, NB50, 9500 Euclid Avenue,Cleveland, Ohio 44195. E-mail: misras@ccf.org. Richard C.Page; Department of Chemistry and Biochemistry, Miami Uni-versity, 651 East High Street, Oxford, Ohio 45056. E-mail:pagerc@miamioh.edu.

Published by Wiley-Blackwell. VC 2014 The Protein Society PROTEIN SCIENCE 2014 VOL 23:833—842 833

homologous to those of the Escherichia coli Hsp70

chaperones DnaK and HscA than to other mam-

malian Hsp70-family members, suggesting that it

may descend from a DnaK-like chaperone of an

endosymbiont precursor of mitochondria. Mortalin

regulates protein folding and quality control in

the mitochondrial matrix,5 as well as iron-sulfur

cluster-biogenesis and insertion into Fe-S apopro-

teins.6 Mortalin participates directly in mitochon-

drial protein import through the TIM (translocase

of the inner membrane) protein complex.7,8 Mor-

talin also cooperates with mitochondrial homeo-

stasis factors including TRAP-1 and the

mitochondrial voltage-dependent anion channel

(VDAC),9 and regulates mitochondrial properties

including ATP levels, membrane potential and

permeability, and response to reactive oxygen

species.10,11

Interestingly, a fraction of Mortalin localizes to

the cytosol where it associates with proteins

involved in signaling, apoptosis, or senescence. Sig-

nificantly, Mortalin binds to cytoplasmic p53 and

negatively regulates this crucial tumor suppressor,

especially under modest stresses.12,13 Mortalin lev-

els and Mortalin-p53 association are increased in

cancer cell lines and tumor models.14–16 Mortalin

prevents nuclear translocation of p5317 and also

blocks p53-mediated suppression of centrosome

duplication.18

As it acts on a variety of binding partners and

is important for mitochondrial homeostasis, Mortalin

is of interest as a therapeutic target in a number of

pathologies. In particular, Mortalin may play a gen-

eral antiapoptotic role in cancers. Elevated levels of

Mortalin are present in individuals with chronic

myelogenous leukemia, colerectal adenocarcinoma,

and hepatocellular carcinoma as well as in several

tumor cell lines.19,20 Mortalin function and levels

are also altered in several neurodegenerative dis-

eases characterized by neuronal mitochondrial dys-

function, including Alzheimer’s and Parkinson’s

diseases.11,21 Such findings have driven efforts to

modulate Mortalin function using small-molecule- or

RNA silencing-based therapeutic approaches.22,23

MKT-077, a rhodacyanine dye, was characterized as

an inducer of mitochondrial toxicity with antitumor

activity well before Mortalin was identified as one of

its targets. MKT-077 induces senescence in cancer

cell lines,24 attenuates Mortalin’s inhibition of

p53,16,25 and may also sensitize cancer cells to

destruction by complement in a Mortalin-dependent

fashion.26 Although Phase I clinical trials of MKT-

077 against advanced solid tumors were halted due

to excessive renal toxicity,27,28 this compound contin-

ues to hold interest as a starting point for inhibitors

of Mortalin and other Hsp70-family members.

Like other Hsp70-family members, Mortalin

consists of a �42 kD NBD and a �25 kD substrate-

binding domain (SBD). The SBD itself is divided

into a b-sandwich domain (SBDb) and a �12 kD a-

helical “lid” domain (SBDa). SBDb contains the sub-

strate binding site with specificity for mixed basic-

hydrophobic sequences. SBDa consists of five helices

and covers the peptide binding site in the high-

substrate-affinity ADP-bound conformation of Hsp70-

family members. In this configuration, the NBD and

SBD do not interact with each other but are tethered

by a short interdomain linker. On ADP-ATP

exchange, the SBDa undergoes a large conforma-

tional change and reorientation, leaving the peptide

binding site open. This conformational change is

accompanied by docking of the interdomain linker

and the SBDb to the NBD, as well as conformational

change in the SBDb, promoting substrate protein

release. Together, these mechanisms underlie

nucleotide-dependent allosteric regulation of the

activity of Hsp70-family members.29

Although a crystal structure of the Mortalin

SBDb with the first two helices of the SBDa was

recently solved (PDB ID 3N8E), no structural data

on the Mortalin NBD has been available. The NBD

is a potential target for nucleotide-competitive

inhibitors as well as potentially allosteric inhibitors

such as MKT-077.30 Elucidation of the NBD struc-

ture may aid development of more specific and

effective mortalin-targeting compounds and is nec-

essary to understand the functional mechanisms of

the full-length protein. In this study, we present

the crystal structure of the NBD of human mortalin

at 2.8-A resolution. We investigate how several resi-

dues in the nucleotide-binding pocket that are not

conserved in other mammalian Hsp70-family mem-

bers affect nucleotide binding. We also model the

interaction of the Mortalin-NBD with its nucleotide

exchange factor mtGrpEL1 and with a potential

Mortalin-binding site of p53. Finally, we suggest

how MKT-077 may bind to the NBD to inhibit Mor-

talin function.

Results and Discussion

Structure of the Mortalin-NBDWe crystallized and solved the structure of the apo-

(NBD of human mortalin to 2.8 A resolution by

molecular replacement, using the NBD of human

Hsp70 (PDB ID 3ATU) as a search model.31 Data

processing and refinement statistics are shown in

Table I. The crystallized protein construct includes

Residues 52–431, containing the entire globular

NBD but not the N-terminal mitochondrial import

sequence (Residues 1–46) or the NBD-SBD interdo-

main linker (Residues 432–441). As in other Hsp70

chaperones, the mortalin NBD is subdivided into

four subdomains [IA, IB, IIA, and IIB, shown in Fig.

1(A)], with the nucleotide-binding pocket located at

the center of the domain. Residues 54–429 are

834 PROTEINSCIENCE.ORG Structure of Mortalin Nucleotide-Binding Domain

resolved fully with the exception of Residues 335–

337, which form the tip of a hairpin loop in subdo-

main IIB.

NBD structures are characterized by conforma-

tional flexibility and variable orientations of the sub-

domains relative to each other, depending on

nucleotide occupancy and binding to cochaperone

proteins.29 Based on a DALI database search,32 our

Mortalin NBD structure most closely resembles

Hsp70-, Hsc70-, and Grp78-NBD structures bound

to ATP or ADP-Pi (backbone Rmsd< 1.4 A over 370

residues). These structures all exhibit a “closed” con-

formation in which subdomains IB and IIB contact

each other. Closed conformations are typically stabi-

lized by ATP or ADP-Pi, which bridge loops from all

four subdomains. However, our Mortalin-NBD con-

struct was purified in a nucleotide-free form; water

molecules and a sodium ion occupy the nucleotide-

binding pocket. The closed conformation of the

Mortalin-NBD is likely induced by extensive crystal

packing and by the water and ion network, which

bridges subdomains IA and IIA. In addition, a salt

bridge between K106 and E313 above the

nucleotide-binding pocket bridges the cleft between

subdomains IB and IIB. A similar salt bridge is

observed in closed conformations of other Hsp70-

family NBDs.33

Interactions at the top of the cleft between sub-

domains IB and IIB also stabilize the closed confor-

mation [Fig. 1(B)]. Aliphatic portions of the V110

and T111 sidechains on subdomain IB interact with

M303, L305, and aliphatic portions of N302, Q306,

and R309. The Q306 sidechain interacts with oppos-

ing backbone amides and stabilizes a helical turn in

subdomain IB that orients V110 and T111. These

complementary regions are less conserved relative to

other eukaryotic Hsp70-family members and the

interactions are more extensive than in other closed

Figure 1. (A) Overall structure of the NBD of human Mortalin.

The four distinct subdomains are labeled and individually col-

ored. The dashed segment represents a short loop that was

not resolved in the electron density map. Waters and a

sodium ion in the nucleotide-binding pocket are shown as

red and yellow spheres, respectively. The dotted box marks

the region shown in panel (B). (B) Closeup view of packing

interactions between subdomains IB and IIB at the top of the

interdomain cleft. Dashed lines indicate putative hydrogen

bonds. The secondary structure cartoon is rendered transpar-

ent for clarity. (C) Comparison of the nucleotide-binding

pocket of Mortalin with that of the Hsp70-NBD with ADP-Pi

(PDB ID 3ATU).31 Mortalin residues are colored as in panel

(A), while Hsp70 residues are colored with white carbons.

Hsp70 residues are labeled in bold italics while plain labels

designate the Mortalin counterparts. In this representation,

the rotamers of several Mortalin residues have been adjusted

(from their orientations in the nucleotide-free crystal structure)

to match the corresponding Hsp70 residues. Use this link to

access the interactive version of this figure.

Table I. Data Collection and Refinement Statistics

Data collection (Beamline ALS 4.2.2)

Wavelength (A) 1.00Space group P212121

Unit cellLengths (A) a 5 51.20, b 5 67.81,

c 5 120.77Angles (�) a5 b 5 c 5 90�

Resolution (A)a 60.38–2.80 (2.90–2.80)Total/unique reflections 10669/1052Completeness (%)a 98.0 (96.2)<I/rI>a 22.6 (4.3)Rmerge

a,b 0.091 (0.383)Refinement statistics

Resolution (A) 60.38–2.80No. of reflections 10008Rwork/Rfree 0.218/0.271No. of atoms/averageB-factor (A2)

2787 (37.9)

Protein 2779 (38.0)Nonprotein 8 (30.6)

R.m.s. deviation bonds (A) 0.013R.m.s. deviation angles (�) 0.86

Ramachandran statistics (%)Favored 96.5Allowed 100.0Disallowed 0.0

MolProbity validation:Clash score/percentile 22.7 (85th percentile,

N 5 141, 2.8 6 0.25 A)MolProbity score/percentile 2.08 (99th percentile,

N 5 4482, 2.8 6 0.25 A)

a Values in parentheses are for the highest-resolution shell.

b Merging R factor is defined as Rmerge 5

Phkl

PijIi hklð Þ2I hklð ÞjP

hkl

PiIi hklð Þ

This figure also includes an iMolecules 3D interactive version

that can be accessed via the link at the bottom of this figure’s

caption.

Amick et al. PROTEIN SCIENCE VOL 23:833—842 835

structures of eukaryotic Hsp70-family NBDs. They

instead resemble the equivalent regions of bacterial

Hsp70s (DnaK). The extensive nature of the interac-

tions between subdomains IA and IIA may affect the

dynamics of opening and closing of the NBD and the

intrinsic rate of nucleotide dissociation, which is 1–2

orders of magnitude less for E. coli DnaK than for

eukaryotic Hsc70.34

Interactions of Mortalin-NBD with nucleotidesThe nucleotide-binding pocket is largely conserved

between Mortalin and other Hsp70-family members,

allowing us to model bound ADP-Pi [Fig. 1(C)].

Exceptions to the overall conservation in the binding

pocket include M389 (Q and S in E. coli DnaK and

human Hsp70, respectively), which would interact

with the side of the adenine moiety; C317 (Arginine

in Hsp70), which packs against the top of the ade-

nine and may form a polar interaction with the ade-

nine N7 atom; and N64 (a tyrosine in Hsp70), which

interacts with the a-phosphate. These sequence dif-

ferences in the nucleotide-binding pocket consis-

tently differentiate mitochondrial Hsp70s from the

eukaryotic cytosolic Hsp70s. In particular, N64 and

M389 are conserved in Mortalins from protists and

fungi to mammals (Supporting Information Figs. S1

and S2). C317 is replaced by isoleucine in some Mor-

talins; the E. coli DnaK equivalents of C317 and

N64 are also isoleucine and asparagine, highlighting

Mortalin’s evolutionary relationship to the prokary-

otic chaperones. M389 appears to be specific to the

mitochondrial Hsp70s and is not conserved in other

Hsp70 families, including endoplasmic reticulum-

resident Hsp70 (Grp75/BiP/HSA5) and other cyto-

solic subfamilies (data not shown).

To explore the functional significance of the

sequence differences in the nucleotide-binding

pocket, we compared the nucleotide affinities of the

Mortalin- and human Hsc70-NBDs. Isothermal titra-

tion calorimetry (ITC) measurements of ADP 1 Pi

and ADP binding to the Mortalin-NBD (Table II;

Supporting Information Fig. S3) show that its affin-

ity is weaker than that of Hsc70. We also measured

the affinity of a Mortalin-NBDN64Y/C317R mutant, in

which Mortalin residues were replaced by their

Hsp70/Hsc70 counterparts. Mortalin-NBDN64Y/C317R

bound ADP 1 Pi even more weakly than wild-type

Mortalin-NBD and ADP binding was too weak to

quantitate by ITC. A Mortalin-NBDN64Y/C317R/M389S

triple mutant was unstable and expressed poorly in

E. coli and we were unable to test it comprehen-

sively. However, a small amount of the purified tri-

ple mutant exhibited similarly low affinity for

ADP 1 Pi as the double mutant in ITC experiments

(data not shown). Similarly to Hsp70/Hsc7031 and

consistent with our crystal structure, phosphate (Pi)

increased the affinity of ADP for Mortalin-NBD or

for Mortalin-NBDN64Y/C317R. We also measured the

affinity of hydrolysis-defective Hsc70-NBD and

Mortalin-NBD mutants35 for ATP. It should be noted

that Hsc70-T204 (and presumably Mortalin-T249)

interact with the g-phosphate of ATP36 and their

mutation likely leads to a lower affinity for ATP

than that of the corresponding wild-type proteins.

Nevertheless, the affinities of Mortalin-NBDT249A

and Mortalin-NBDN64Y/C317R/T249A for ATP were also

substantially weaker than that of Hsc70-NBDT204A

(Table II; Supporting Information Fig. S3).

The nucleotide affinity is, thus, not determined

solely by the proximal residues of the nucleotide-

binding pocket. Conformational dynamics of the

respective NBDs, which are influenced by segments

outside the nucleotide-binding pocket, likely influ-

ence their effective nucleotide affinities and dissocia-

tion/exchange rates.1,34 Recent studies showed that

full-length Ssc1, a Mortalin orthologue in Saccharo-

myces cerevisiae, fluctuates between nucleotide-free

and bound states even in the presence of substantial

concentrations of ATP or ADP, and also hint that

the overall nucleotide affinity is lower in Mortalin

than in other Hsp70 family members.37 A structure-

based alignment of the Hsp70, Mortalin, and DnaK

NBD sequences shows nonconserved residues that

mediate intersubdomain contacts or are located in

hinges between the subdomains (Supporting Infor-

mation Fig. S2). These residues may contribute to

differences in conformational dynamics between the

Hsp70 and Mortalin-NBDs.

Disease-associated mutation on the surface of

Mortalin-NBD

Mitochondrial dysfunction is a general hallmark of

dopaminergic neurodegeneration in Parkinson’s dis-

ease. Genetic studies identified three Mortalin

mutants in Parkinson’s disease patients: P509S and

A476T in the SBD, and R126W in the NBD.11,38 The

R126W mutation may promote aggregation in vivo

Table II. Nucleotide Affinities of the Mortalin- andHsc70-NBDs

Nucleotide Protein Kd (mM)

ADP-Pi Hsc70-NBD 0.029 6 0.002Mortalin-NBD 5.3 6 0.3Mortalin-NBDN64Y/C317R 16 6 3

ADP Hsc70-NBD 0.052 6 0.02Mortalin-NBD 620 6 110Mortalin-NBDN64Y/C317R n.d.b

ATP Hsc70-NBDT204A 3.9 6 0.7Mortalin-NBDT249A 29 6 7Mortalin-NBDN64Y/C317R/T249A n.d.b

a Dissociation constants (Kd) measured by ITC; see Sup-porting Information Figure S2 for raw ITC data and titra-tion curves.b ITC signal too weak/noisy to properly calculate dissocia-tion constants. We estimate that the Kd values are in themillimolar range for these measurements.

836 PROTEINSCIENCE.ORG Structure of Mortalin Nucleotide-Binding Domain

leading to loss of mortalin function.39 R126 lies on

the surface of the NBD at the interface between sub-

domains IIA and IA (Fig. 2). Interestingly, only a

minority of Trp rotamers are accommodated at this

position, and even these require movement of the

nearby sidechains of Q201 and D130. Replacement

of R126 by tryptophan and local remodeling may

create a more acidic surface patch (Fig. 3), or the

poor packing of the Trp may destabilize the Mortalin

fold. Either of these alterations could contribute to

aggregation and suppression of function of the

mutant Mortalin protein.

Model of interaction between Mortalin-NBD and

mitochondrial GrpEL1Mortalin interacts with a mitochondrial J-domain

protein Tid-1, the cochaperone Hep, and two

potential nucleotide exchange factors in mitochon-

dria.40–42 In analogy with other Hsp70-family mem-

bers, these cochaperones accelerate the low intrinsic

rates of ATP hydrolysis and ADP/ATP exchange,

respectively, and are essential for effective chaper-

one function. Further emphasizing the connection

between Mortalin and prokaryotic Hsp70s, the

nucleotide exchange factors are homologous to

GrpE, the nucleotide exchange factor for DnaK.

Termed GrpELike1 and 22 (GrpEL1 and GrpEL2),

they are approximately 43% identical and 65% simi-

lar to each other. We generated a model of the

Mortalin-NBD in complex with a homology model of

human GrpEL1 (Fig. 4), based on structures of

E. coli and Geobacillus kaustophilus DnaK:GrpE

complexes.43,44 We modeled the Mortalin-NBD in an

open conformation, which is characteristically

induced by GrpE on DnaK, by tilting subdomain IIB

away from subdomain IB about the IIA/IIB hinge.

GrpEL1 has moderate homology to bacterial GrpEs

(�25% identical and �45% similar). However, key

interactions are conserved; the helical portion of the

asymmetric GrpEL1 dimer “head” primarily contacts

subdomain IIB while the extended wing of one of

the GrpEL1 protomers contacts subdomain IB (Fig.

4). The coiled-coil “tail” of the GrpEL1 dimer also

makes contacts with subdomain IA. The putative

contact residues of GrpEL1 and Mortalin are shown

in closeups in Supporting Information Figure S4.

Interaction of Mortalin-NBD with p53 and

MKT-077

Several studies have probed the interaction of Mor-

talin with p53 and obtained evidence for a nonca-

nonical substrate recognition mode that differs from

the well-understood binding of substrates to the

SBDs of Hsp70-family chaperones. Deletion analysis

identified a putative p53-interacting region on the

Mortalin NBD, encompassing Residues 253–282.45

These residues define a surface formed by two b-

strands and a hairpin turn in subdomain IIA, and

extend into a helix from subdomain IIB. One of

these strands serves as a docking site for the NBD-

SBD interdomain linker in the putative ATP-bound

form of Mortalin, in analogy with the structure of

ATP-bound DnaK.46,47 A segment from the Mortalin

interdomain linker (434DVLLLDV440) is identical to

the equivalent segment in DnaK and likely forms an

additional parallel beta strand to dock against sub-

domain IIA [Fig. 4(A)]. Interestingly, a comparable

sequence (323LDGEYFTLQIRGRE337) is present

Figure 2. Location and effect of the Parkinson’s disease

mutation R126W. (A) Location of R126 mapped onto the

Mortalin-NBD surface. NBD subdomains are colored and

labeled as in Figure 1. Location of R126 and the nearby D130

and Q201 residues are shown as a white patch and marked

by a dashed oval. (B) Surface electrostatic potential of the

R126W mutant calculated using APBS,62 within a range of

25(red) to 15kT (blue). The dashed oval marks the location

of W126, D130, and Q201. (C) Surface electrostatic potential

of wild-type Mortalin-NBD, colored and marked as in panel

(B). Use this link to access the interactive version of this

figure.

Figure 3. Homology model of the Mortalin-NBD in complex

with mitochondrial GrpEL1. Mortalin-NBD subdomains are

colored as in Figure 1. The protomers of the GrpEL1 dimer

are colored yellow and orange. Detailed views of the interac-

tions are shown in Supporting Information Figure 4.

This figure also includes an iMolecules 3D interactive version

that can be accessed via the link at the bottom of this figure’s

caption.

Amick et al. PROTEIN SCIENCE VOL 23:833—842 837

near the tetramerization domain of p53. A peptide

mimic of this sequence disrupts Mortalin:p53 inter-

action.48 These residues form a flexible linker

between the tetramerization helix and the preceding

core/DNA-binding domains of p53. We docked resi-

dues from this sequence (324DGEYFTLQI332), mim-

icking the docking of the Mortalin interdomain

linker [Fig. 5(B)], to model how the Mortalin-NBD

may interact with p53. This model does not conflict

with or exclude the reported binding of the

Mortalin-SBD to other regions of p53 such as the

p53 negative regulatory domain.49 A recent in vitro

study suggests that Mortalin Residues 260–288 on

the NBD may form another binding site for the p53

C-terminal negative regulatory domain.50 In addi-

tion, the association between Mortalin and p53 may

be regulated by Mortalin’s J-domain cochaperone

Tid1, which also binds to p53.51 The Mortalin NBD

region encompassed by Residues 253–282 includes a

potential interaction site for the J-domain of Tid1,53

suggesting that Tid1 could pass p53 onto the Mor-

talin SBD while itself docking onto the Mortalin

NBD.

The putative binding site for the rhodacyanine

dye MKT-077, which downregulates p53 sequestra-

tion by Mortalin, was mapped to Mortalin Residues

252–310, overlapping with the putative p53- and/or

Tid-1-interacting sites of the Mortalin NBD.25 A

binding site for MKT-077 on human Hsc70 has been

proposed based on NMR and computational docking

data.30 Based on these studies, we modeled MKT-

077 sandwiched between Mortalin Residues 267–271

and the sidechain of Y196 [Fig. 5(C)]. At this posi-

tion, the MKT-077 may disrupt p53 docking by per-

turbing the conformation of the interacting Mortalin

b-strand (Residues 260–270). As the Y196-

containing loop likely approaches Residues 267–271

in the ATP-bound conformation of Mortalin,46,47

MKT-077 may also destabilize the ATP-bound con-

formation and indirectly prevent the association of

Tid1. Thus, there are several potential ways for

MKT-077 to both block Mortalin function generally

and to specifically prevent the association between

Mortalin and p53.

Conclusions

Together with the recent structure of the Mortalin-

SBD (PDB ID 3N8E), our crystal structure of the

NBD confirms the overall structural similarity

between Mortalin to the prokaryotic DnaK chaper-

ones and the diversified eukaryotic Hsp70-family

members. Structural characterization of full-length

Mortalin in the nucleotide-free, ATP-, and ADP-

bound states would be a valuable next step in char-

acterizing this important chaperone. Our results

establish a structural basis to elucidate the interac-

tions of Mortalin with mitochondrial cochaperones,

with the mitochondrial import machinery and with

Figure 4. Models of Mortalin-NBD binding to p53 and MKT-077. The Mortalin NBD is colored and labeled as in Figure 1. The

NBD secondary structure cartoon is shown partly transparent for clarity. (A) Model of the interaction between the Mortalin

NBD-SBD interdomain linker sequence (shown as white stick model with italicized labels) and the b-sheet of subdomain IIA.

The model was constructed based on the ATP-bound structure of DnaK (PDB IDs 4B9Q, 4NJ4).46,47 (B) Interaction between

p53 sequence located N-terminal to the p53 tetramerization domain and the same b-strand as in panel (A). The p53 sequence

is shown as yellow sticks and labeled in yellow italics. (C) Five high-scoring poses of MKT-077 docked to the Mortalin-NBD.

MKT-077 models are shown as thick sticks. The inset shows the structure of MKT-077 (no hydrogens are shown). In the

docked orientations, the methyl-benzothiazoline moiety of MKT-077 is closest and the ethylpyridine moiety is furthest from the

viewer. Selected Mortalin residues within 4 A of MKT-077 are shown and labeled. The region of the NBD shown corresponds to

the upper portion of the regions shown in panels (A and B).

838 PROTEINSCIENCE.ORG Structure of Mortalin Nucleotide-Binding Domain

substrates both within mitochondria and in the cyto-

sol. Finally, the NBD structure may inform the

design of more potent and specific Mortalin-

targeting compounds, including direct competitors of

ATP as well as allosteric compounds that function

similarly to MKT-077.30 Differential and context-

specific targeting of Mortalin offers a potentially

important therapeutic avenue for the treatment of

cancers, neurodegenerative diseases, or other pathol-

ogies characterized by mitochondrial dysfunction.

Materials and Methods

Cloning, expression, and purification ofMortalin-NBD

A pET281-Mortalin plasmid containing the coding

sequence for human Mortalin Residues 52–679) was

used as a template.54 The Mortalin NBD sequence

was amplified by polymerase chain reaction and

inserted into the pGST||2 expression vector.55 The

resulting plasmid, pGST||2-Mortalin(52–431), codes

for an N-terminal glutathione-S-transferase tag, fol-

lowed by a tobacco etch virus (TEV) protease cleav-

age site and human Mortalin Residues 52–431.

Sequence-verified constructs were transformed into

E. coli Rosetta2 (DE3) cells (EMD Chemicals) for

expression.

The GST-tagged Mortalin NBD was expressed

in Luria-Bertani media induced with 0.25 mM iso-

propyl b-d-1-thiogalactopyranoside and shaken for

20 h at 20�C. Cultures were harvested by centrifuga-

tion and resuspended in 20-mM Tris-HCl pH 8.0, 50-

mM NaCl, 5-mM 2-mercaptoethanol, 1 mg/mL lyso-

zyme (Sigma), 20 mg/mL DNaseI (MP Biomedicals),

100 mg/mL 4-(2-Aminoethyl) benzenesulfonyl fluoride

hydrochloride (Gold Biotechnology). Resuspended

cells were flash-frozen, thawed, and lysed overnight

at 4�C. Lysates were clarified by centrifugation,

loaded onto a glutathione Sepharose column (GE

Healthcare) equilibrated in 50 mM NaCl, 20 mM

Tris-HCl pH 8.0, 5 mM 2-mercaptoethanol (buffer

A), and washed with 15 column volumes of buffer A.

The Mortalin-NBD was cleaved on the column with

0.75 mg of His6-tagged TEV protease56 overnight. A

5-mL HisTrap HP column (GE Healthcare) and an

additional glutathione Sepharose column were added

to bind the TEV protease and cleaved GST. The col-

umns were washed with three column volumes of

buffer A, and the flow through containing Mortalin

NBD was collected. The flow through was dialyzed

into 20-mM Tris-HCl pH 7.7, concentrated by cen-

trifugation and applied to a Superdex-70 16/60 size

exclusion column. The protein was eluted in the

same buffer and peak fractions were applied to a

HiTrap Blue HP column (GE Healthcare), washed

with five column volumes of 20 mM Tris-HCl pH 7.7

and eluted with a 10 column volume gradient of 0–2

M NaCl, 20-mM Tris-HCl pH 8.0. The peak fractions

were pooled and dialyzed into 50 mM NaCl, 20 mM

Tris-HCl pH 8.0.

Mortalin-NBD point mutants were generated

from the wild-type construct by polymerase chain

reaction using the Quikchange-II site-directed muta-

genesis kit (Agilent) and verified by sequencing.

Mortalin-NBD mutants were expressed and purified

in the same manner as the wild-type protein con-

struct. Wild-type and mutant Hsc70-NBD (human

Hsc70 Residues 1–384) were cloned into the

pHis||2 vector,55 expressed as 6xHis-tagged con-

structs and purified on a His-Trap HP Sepharose

column (GE Healthcare), followed by Superdex-70

size exclusion and HiTrap Blue purification steps as

described above.

Crystallization and structure determinationFresh Mortalin-NBD was concentrated to 19 mg/mL

by ultracentrifugal filtration and used for sitting

drop crystallization trials, carried out with a Gry-

phon crystallization robot (Art Robbins Instru-

ments). Sitting drops contained 0.2 mL of protein

and 0.2 mL of well solution. Initial screening was

carried out using the sparse matrix crystallization

screens JCSG Core I-IV (Qiagen) and MCSG 1–4

(Microlytic). Optimizations of initial hits from the

sparse matrix screens identified an optimal reservoir

solution of 0.2 M di-ammonium tartrate and 20%

PEG 3350. Crystals were cryoprotected by brief

transfer through LV CryoOil (MiTeGen) and immedi-

ately frozen in liquid nitrogen.

X-ray diffraction data were collected at 1.00 A on

beamline 4.2.2 at the Advanced Light Source, Law-

rence Berkeley National Laboratory and processed

using XDS.57 Phases were calculated by molecular

replacement using the PHASER58 component of

PHENIX.59 The structure of human Hsp70 in the

ADP- and Mg-bound state (PDB ID 3ATU) was uti-

lized as the search model.31 Automated rebuilding of

the molecular replacement solution in PHENIX Auto-

Build60 allowed for placement of human Mortalin

Residues 54–334 and 337–429. The rebuilt model was

subjected to iterative rounds of model building in

Coot61 and refinement in PHENIX. Atomic coordi-

nates and structure factors have been deposited in

the Protein Data Bank (PDB ID 4KBO). Stereochemi-

cal and geometric analyses of the Mortalin-NBD

structure were conducted with MolProbity.62 All

molecular structure figures were prepared with

PyMOL.63 Surface electrostatics shown in Figure 2

were calculated using the APBS64 plugin for PyMol.

Isothermal titration calorimetryCalorimetric measurements were performed at 25�C

using MicroCal iTC200 or VP-ITC isothermal titra-

tion calorimeters (MicroCal, Northampton, MA).

Proteins were dialyzed against 20 mM Tris, 50 mM

NaCl, 5 mM KCl, 5 mM MgCl2, pH 8.0. Protein

Amick et al. PROTEIN SCIENCE VOL 23:833—842 839

concentrations were determined using a 660 nm Pro-

tein Assay (Pierce) and were 20–150 mM. Nucleo-

tides were freshly dissolved in the above buffer at

500 mM to 3 mM concentration and, if needed, the

pH was adjusted to 8.0 with NaOH. For ADP 1 Pi

binding measurements, the protein and nucleotide

buffers also contained 5-mM Na2HPO4. Binding iso-

therms were corrected by subtracting results from a

control experiments with nucleotide injected into

buffer. Dissociation constants (Kd) were estimated

by nonlinear least-squares fitting to a single-site

binding model using Origin v7.0 (OriginLab).

Homology and computational modelingTo model the Mortalin-NBD complex with GrpEL1,

Mortalin-NBD subdomains were first aligned to

their DnaK-NBD counterparts within the G. kausto-

philus DnaK/GrpE complex structure44 (PDB ID

4ANI). A GrpEL1 homology model was generated

using the I-Tasser web interface,65 which yielded a

very good-quality model (C-score 5 1.38,

Rmsd 5 2.3 6 1.7 A). Peptide bonds linking the sub-

domains were minimally rebuilt using Coot61 to

ensure realistic stereochemistry. The reconstructed

Mortalin-NBD/GrpEL1 complex was subjected to 10

individual rounds of all-atom refinement using

Rosetta relax.66,67 The lowest total Rosetta energy

score (total_energy) decoy from the family of 10 mod-

els was chosen as the representative model.

To model the p53 interaction with the Mortalin-

NBD, an initial docking pose for human p53 Resi-

dues 324–332 was modeled in Coot61 utilizing struc-

tures of ATP-bound DnaK (PDB IDs 4B9Q and

4JN4) as templates.46,47 The initial docking pose

was prepacked with Rosetta68 to produce a starting

model for Mortalin-NBD/p53 Rosetta FlexPepDock

calculations.69 A family of 500 decoys were gener-

ated by Rosetta FlexPepDock using extra Chi1

rotamers, extra aromatic-Chi2 rotamers, and

backbone-backbone hydrogen bond distance con-

straints to maintain the b-sheet hydrogen bonding

pattern between p53 Residues E326–F328 and Mor-

talin Residues F262–V264. The decoy with the low-

est Rosetta interface score (I_sc) was used as a

representative model.

To dock MKT-077 to the Mortalin-NBD, an ini-

tial docking pose was prepared by aligning the

Mortalin-NBD to the yeast Hsc70-NBD utilized in

previously described Hsc70/MKT-077 AUTODOCK

calculations.30 The best MKT-077 docking pose iden-

tified in this study was added to the aligned

Mortalin-NBD and prepacked with Rosetta68 to gen-

erate the starting model. Ligand geometry and

charge were optimized using PHENIX eLBOW70 and

used to prepare the Rosetta ligand parameter file for

MKT-077. RosettaLigand71 docking calculations

allowed for backbone and side-chain flexibility with

extra Chi1 and aromatic-Chi2 rotamers for

Mortalin-NBD and full flexibility for MKT-077 with

permitted ligand translations away from the starting

pose of up to 65 A along the x, y, and z axes. An

ensemble of 10,000 decoys was generated and the

top five percent were clustered based on overall

Rosetta energy score (total_score). A representative

family of models was selected from the top five per-

cent cluster as the five decoys with the lowest

Rosetta interface score (I_sc).

Acknowledgments

The authors acknowledge Drs. Satya Yadav and

Smarajit Bandyopadhyay and the Molecular Bio-

technology Core of the Lerner Research Institute

(Cleveland Clinic) for access to and assistance with

isothermal titration calorimetry instrumentation.

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842 PROTEINSCIENCE.ORG Structure of Mortalin Nucleotide-Binding Domain