structural insights into the interaction of rocki with the switch

Structural insights into the interaction of ROCKI with the switch regions of RhoA*

Radovan Dvorsky, Lars Blumenstein, Ingrid R. Vetter, and Mohammad Reza Ahmadian

Max-Planck-Institute fuer molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-

Hahn-Strasse 11, 44227 Dortmund, Germany

Correspondence should be addressed to M.R.A:

Phone: +49 231 1332105; Fax: +49 231 1332199

E-mail: [email protected].

*This work was supported in part by a Marie Curie Fellowship by the European Community

(R.D.) and by the Deutsche Forschungsgemeinschaft (L.B.).

The coordinates of human GppNHp-bound RhoA in complex with the Rho-binding domain of

ROCK I have been deposited in the Protein Data Bank (accession code xxxx).

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 2, 2003 as Manuscript M311911200 by guest on M

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The Rho-ROCK pathway modulates the phosphorylation level of a variety of important

signaling proteins and is thereby involved in miscellaneous cellular processes including

cell migration, neurite outgrowth and smooth muscle contraction. The observation of

the involvement of the Rho/ROCK pathway in tumor invasion and in diseases such as

hypertension and bronchial asthma make it an intersting target for drug development.

We herein present the crystal structure of the complex between active RhoA and the

Rho-binding domain (RBD) of ROCKI. The RBD structure forms a parallel alpha-

helical coiled-coil dimer and in contrast to the published Rho-PKN structure binds

exclusively to the switch I and switch II regions of the GppNHp-bound RhoA. The

switch regions of two different RhoA molecules form a predominantly hydrophobic

patch which is complementary bound by two identical short helices of 13 residues (aa

998-1010). The identified ROCK-binding site of RhoA strikingly supports the

assumption of a common consensus binding site for effector recognition.

Running title: Rho-ROCK structure

Keywords: Coiled-coil, effector, PKN, PRK, Rho, Rho kinase, Rho binding domain, ROCK,

small GTPase.

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INTRODUCTION

A hallmark of small GTPases is their ability to undergo structural changes in response to

binding of GDP or GTP. The inactive GDP-bound and the active GTP-bound state are

recognized by different partner proteins, thereby allowing the small GTPases to function as

molecular switches (1, 2). Nucleotide binding, hydrolysis and localization are regulated by

three families of regulatory proteins: guanine nucleotide exchange factors (GEFs) (3-5),

GTPase activating proteins (GAPs) (6) and guanine nucleotide dissociation inhibitors (GDIs)

(7). The GTP-bound state provides a platform for the selective interaction with downstream

targets, the so-called effector proteins.

The Rho subfamily of small GTPases regulates diverse cellular processes via their specific

effector proteins which are either serine/threonine protein kinases such as ROCK, PKN/PRK1

and citron kinase or scaffold proteins such as rhophilin, rhotekin, citron and diaphanous

(reviewed in 8-10). Among the best-characterized Rho effectors are ROCK proteins. Two

ROCK isoforms, ROCKI/ROKβ/p160ROCK and ROCKII/ROKα/Rho kinase that share 65%

overall identity and 95 % homology have so far been identified (11-14). They were first found

as mediators of stress fibers and focal adhesion formation (15), but further studies revealed

that ROCK is involved in many other cellular processes including smooth muscle contraction,

cell migration and neurite outgrowth (16,17). Interest in the Rho-ROCK pathway emerges

from the fact that abnormal activation of this pathway plays a role not only in tumor invasion

and metastasis (18-20) but also in diseases such as hypertension and bronchial asthma

(21,22).

ROCK proteins consist of an N-terminal kinase domain followed by a central putative coiled-

coil region, a pleckstrin homology domain and a cysteine rich domain at the C-terminus

(11,12,23; Fig. 1A). The Rho-binding domain (RBD) within the predicted amphipathic α-

helical coiled-coil (Fig. 1A) is responsible for the recognition and binding of the active Rho


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proteins (13). Coiled-coil structures have also been predicted for the RBDs of most Rho

effectors (rhotekin, rhophilin, Citron kinase and Kinectin) (9). The most recent crystal

structure of bovine ROCKII showed that two long helical strands of the RBD form a parallel

coiled-coil dimer (24). A different scenario has been described for the PKN, where two

adjacent domains at the N-terminus of PKN, called homology region (HR)1a and HR1b, have

been identified to bind RhoA (25,26). Both domains form anti-parallel coiled-coil (ACC)

structures called ACC finger (27,28). HR1a of PKN binds RhoA at two different contact sites

(I and II) from which only contact site II overlaps with the switch regions (27). Mutagenesis

studies have shown that HR1b is also capable of binding Rac1, another GTPase of the Rho

family, at a region corresponding to the contact site I of RhoA (28).

To examine the interaction of ROCK with RhoA we determined the crystal structure of the

complex between the human ROCKI-RBD (residues 947-1015) and the truncated form of

human RhoA (residues 1-181) bound to the non-hydrolysable GTP-analogue GppNHp.

Whereas the overall structure of the ROCKI-RBD parallel coiled-coil is distinct from the

structure of unbound ROCKII-RBD, the C-terminal part, where the interaction with RhoA

takes place, shares high structural similarity. A short stretch of 13 residues at the C-terminus

creates a minimal Rho interacting motif of ROCK by forming a parallel coiled-coil dimer.

This motif represents a novel type of interaction with a small GTPase by employing both

helices interacting with the switch regions of RhoA in a complementary manner.


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MATERIALS AND METHODS

Cloning, expression and protein purification. — The truncated RhoA (aa 1-181) and ROCKI-

RBD (aa 947-1015) were generated by PCR and cloned into the pGEX-4T1 vector

(Pharmacia) via BamHI/XhoI restriction sites. Recombinant glutathione S-transferase (GST)

fusion proteins were purified by a GSH-Sepharose column (Pharmacia, Freiburg) using 50

mM Tris/HCl pH 7.5, 100 mM NaCl and 5 mM MgCl2 and 5 mM dithio erythritol (DTE).

After cleavage with thrombin proteins were purified using a benzamidine column (Pharmacia,

Freiburg) to remove the protease, and subsequent gel filtration (Superdex G75, Pharmacia,

Freiburg). Purified protein quality and the nucleotide binding capacity were analysed by SDS-

PAGE and reversed-phase HPLC using a C-18 column (ODS-Hypersil, 5 µm, Bischoff,

Leonberg, Germany), respectively. The complex of RhoA·GppNHp and ROCKI-RBD was

isolated by gel filtration (Superdex 75, 16/60, Pharmacia).

Crystallization and data collection. — Crystals of truncated RhoA (residues 1-181) in

complex with the non-hydrolysable GTP-analogue GppNHp and ROCKI-RBD (residues 947-

1015) were grown by vapor diffusion in hanging drops at 20°C by adding equal volumes of

reservoir solution (100mM Tris/HCl pH 7.5, 12% Polyethylene glycol (PEG) 3350, 2%

isopropanol) and protein solution (10mg/ml in 30mM Tris/HCl pH 7.5, 4mM MgCl2 and

2mM DTE). Crystals appeared after 2 days and grew as plates attached to spherulites to a

final dimension of 0.1×0.5×0.05mm in about 10 days. The crystals belong to space group P21

with unit cell dimensions of a = 34.4 Å, b = 89.5 Å, c = 98.1 Å and β = 91.9°. For data

collection at 100 K, the solution containing the crystals was adjusted to 100mM Tris/HCl pH

7.5, 12% PEG 3350, 15% glycerol and 3% isopropanol. Cryo-protected crystals were then

suspended in a rayon loop (Hampton Research) and flash frozen in liquid nitrogen. X-ray

diffraction data were collected on an ADSC Q4 CCD detector at the ID14-1 beam line at the


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European Synchrotron Radiation Facility (ESRF) and processed using the programs DENZO

and SCALEPACK (29). Due to the technical reason the data set was collected only at 2.6 Å

resolution.

Structure determination and refinement. — Initial phases were calculated after molecular

replacement using the program AMoRe (30) with a search model of RhoA based on the

RhoA-PKN structure (27). The Rcryst value was 36% after rigid body refinement performed

with the program CNS (31) and the resultant initial map showed clear electron density for

most of RhoA but only for a part of the RBD molecule. The model was manually built and

refined by using the programs O (32) and CNS (31), respectively. Non-crystallographic

restraints were applied on two RhoA molecules and on the C-terminal parts of the ROCKI

molecules in the asymmetric units. The final model refined to a Rcryst=21.9% and Rfree=25.7 %

contains 69 and 70 residues of the two RBD molecules spanning residues 947-1013 and 947-

1014 (with two additional residues at the N-termini due to the thrombin cleavage), 179

residues of RhoA (residues 3-181), two GppNHp molecules, two magnesium ions and 55

water molecules (Table 1).


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RESULTS AND DISCUSSION

Overall structure. — The asymmetric unit of the crystal contains a α-helical coiled-coil

ROCKI-RBD dimer and two RhoA molecules (Fig. 1B). A two-fold non-crystallographic

symmetry axis runs through the center of the coiled-coil dimer at the interacting interface with

RhoA (Fig. 1C). The structure of RhoA·GppNHp is very similar to the GTPγS-bound RhoA

structure alone (33) (with a root mean square deviation (r.m.s.d.) of 0.42 Å for 181 Cα-atoms)

and in the complex with PKN-RBD (r.m.s.d. of 0.33 Å for 181 Cα-atoms) (27). The binding

of GppNHp and the coordination of the magnesium ion are conserved.

The ROCKI-RBD forms a 95 Å long α-helix which assembles into a parallel coiled-coil (Fig.

1D) with a total buried solvent accessible surface of 3200 Å2. Coiled-coil arrangement of

ROCKI RBD is consistent with the published data on bovine ROCKII RBD (24,34) which

showed that ROCK-RBDs exist as dimers. A periodicity of seven residues over two helical

turns called heptad repeat is the characteristic feature of a canonical left-handed coiled-coil.

These seven residues are commonly designated as abcdefg (35). The interaction between the

helices in a canonical coiled-coil is mediated by the hydrophobic residues at the positions a

and d and oppositely charged residues at the positions e and g (35,36). Seven heptad repeats

were found along the coiled-coil of ROCKI-RBD between the residues I952 and R1012 (Fig.

1D) together with three so called `stutters´ at the residue positions M966, K977 and I981

(marked by red arrows). Stutters are insertions of four residues into the heptad repeat pattern

that cause a non-canonical right-handed twist of a coiled-coil (35). The twist of ROCKI- RBD

is therefore left-handed at the N-terminal and C-terminal part but right-handed in the central

region (residues 965-985) (Fig. 1D). The coiled-coil structure of bovine ROCKII alone (24)

reveals a persistent heptad repeat motif without ´stutters’ forming a canonical left-handed

coiled-coil. Although we cannot exclude that the observed differences in the overall spatial

arrangement compared to the human ROCKI are caused by crystal packing, it is very likely


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that they emerge due to deviations in the N-terminal and central regions of the primary

structure of these isoforms. In contrast to that the C-terminal parts of both structures, where

ROCKI contacts RhoA (see next section), are very similar (Fig. 1, D-F), implicating that this

region is structurally conserved. The repetition of hydrophobic residues at the a and d

positions within the heptad repeat is not strictly conserved in the middle of the ROCKI RBD

and as a consequence the structure is less ordered. It is worth to note that three of four amino

acids at positions e and g in the C-terminal part are hydrophobic and increase the exposed

hydrophobic surface facilitating the interaction with RhoA (see next section).

The RhoA-ROCK-RBD interface. — The interface between the helical ROCKI dimer and one

of the RhoA molecules buries a solvent accessible surface of 1328 Å2, comparable to the

interfaces of Ras-PI3Kγ (37) and Rac-p67phox (38). The structure of the RhoA-ROCKI

complex defines a 13-residue left-handed coiled-coil at the C-terminal part of the

ROCK-RBD (residues 998-1010) as the minimal Rho interacting motif (Fig. 1A). This Rho

binding motif is invariant in all ROCK proteins from different organisms. Both switch regions

of RhoA are involved in the interaction with ROCKI. One of the coiled-coil helices (cyan)

faces switch I whereas the second coiled-coil helix (blue) faces switch II (Fig. 2, B and C).

The interface between the RhoA molecules and the RBD dimer is formed by a combination of

hydrophobic and electrostatic interactions (Fig. 2, B and C). The interaction between the

hydrophobic patches of RhoA (P36, V38, F39, Y66, L69 and L72) and ROCK (A1002,

V1003, L1006, A1007, M1010) is stabilized by a number of electrostatic interactions at their

edge (E40-K1005; D65-K999; R68-N1004). For better visibility, the ROCK residues are

indicated in italics. Two polar residues (Y66 and K1005) also partially contribute to the

hydrophobic interaction while their hydrophilic parts interact with the main chain carbonyl

groups of L998 and F39, respectively. Remarkably, four amino acids of RhoA (V38, F39,


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D65, Y66) interact with both RBD helices and stabilize their dimeric structure (Fig. 2, B and

C). Two key residues (L998 and K1005) of the right helix (cyan) of the ROCK-RBD create a

hydrophobic cluster where L998 tightly binds to a pocket formed by P36, Q63, D65 and Y66

of RhoA whereas K1005 contacts V38 and F39 (Fig. 2, B and C). In addition, K1005,

stabilized by an intramolecular salt bridge with E1008 (Fig. 2A), mediates two electrostatic

interactions with the carboxyl group of E40. Furthermore, K999 and N1004 from the left helix

(blue) of the RBD form hydrogen bonds with D65 and R68 of RhoA, respectively.

Our structural data of the binding interface between RhoA and ROCKI nicely matches with

the mutational analysis of previous studies. Two regions of RhoA (residues 23-40 and 75-92)

have been implicated as binding determinants for ROCKI (39). Fig. 2 B and C shows that the

C-terminal part of the first region overlaps with the switch I region that interacts with ROCKI

RBD. Accordingly, the mutations of F39 and E40 have been shown to disrupt ROCKI binding

(40,41) supporting the crucial role of these residues in the RhoA-ROCKI interaction

described in this study. Unlike the previous study (39) our structure does not confirm a direct

involvement of the second region (residues 75-92) in the ROCK RBD interaction. As the

central part of this region is buried in the hydrophobic pocket of both RhoA and Rac1 proteins

it is very likely that substitutions such as F78I or I80M (Rac-Rho chimera) may cause overall

structural changes creating or disrupting the GTPase-effector interaction. Mutations of

ROCKII at residues K999, Q1001, N1004 and K1005, which are all important components of

the interface of both ROCK helices, resulted in loss of RhoA binding (11). It has also been

proposed by Shimizu et al. (24) that the C-terminal part of the coiled-coil structure of

ROCKII-RBD binds the RhoA molecule. However, the described positive electrostatic

potential of this RBD serves only as a long-range driving force for initial recognition because

the RBD-RhoA-interface is predominantly mediated by hydrophobic interactions (Fig. 3B).


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Comparison with the RhoA-related proteins. — Whereas RhoA has been the most thoroughly

investigated member of the Rho subfamily, the role of the highly related RhoB and RhoC that

share over 85% amino acid identity with RhoA has not been completely elucidated (42). It has

been shown that ROCKII binds to RhoA as well as to its isoforms RhoB and RhoC (11).

Since the region of RhoA contacting ROCK-RBD is identical to the corresponding sequence

of RhoB and RhoC, both of these isoforms are likely to interact with ROCKI in a same way

as RhoA. On the other hand, Rnd proteins, a GTPase deficient Rho-related protein subfamily,

have been shown to antagonize some effects of the RhoA induced cytoskeletal reorganization

(43,44). Although the Rnd3 structure reveals remarkable similarities in the arrangement of the

switch I region it shows major amino acid and surface deviations at the switch II region

compared to RhoA (45,46). The deviation at positions Q63, E64 of D65 of RhoA to SPY in

Rnd3 (45) is apparently the reason why Rnd3 does not bind the ROCKI-RBD (P. Chardin,

personal communication), since both Q63 and D65 of RhoA are essential for the interaction

with ROCKI (Fig. 2, B and C). Thus, a competition between Rnd3 and RhoA for ROCK

binding can be excluded as a possible explanation for the antagonistic effect of Rnd3 on Rho

signaling. Most recently, it has been shown that Rnd proteins bind the N-terminus of ROCKI

close to the kinase domain (47) and thereby prevent Rho-ROCK-interaction. Another model

proposes a downregulation of Rho by activation of Rho-specific GAPs via Rnds (48,49).

ROCK specificity for Rho, versus Rac or Cdc42. — It has been previously shown that

ROCKII binds exclusively RhoA but not Rac1 or Cdc42 (11). The residues involved in

ROCK-RBD binding differ among these GTPases only in one position, namely in E40 of

RhoA which corresponds to D38 in Rac1 and Cdc42. E40 interacts with the K1005 of ROCKI

and stabilizes its orientation, thus facilitating the hydrophobic contacts to V38 and F39 (Fig.

2). Its change to aspartic acid may weaken the interaction with K1005 due to the longer


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distance between the corresponding electrostatic counter ions. To analyze the corresponding

residues on the structural level, we superimposed the GTPγS-bound RhoA (33), the GppNHp

bound Rac1 (50) and the GppNHp bound Cdc42 in complex with RhoGAP (51) with respect

to the ROCK binding regions of RhoA in the presented structure (Fig. 3A). Strikingly, the

peptide backbone and the relative orientation of most side chains are well conserved except

for residues F39, Y66 and L69 which strongly contribute to the extended hydrophobic

interaction with ROCK as described above. A comparison of the hydrophobic patches on the

RhoA, Rac1 and Cdc42 surface shows similarities at positions P36, V38, Y66 and L72 in

RhoA (P34,V36, Y64 and L70 in Rac and Cdc42) (Fig. 3B). However, differences in the

orientation of F39 and L69 in RhoA (F37 and L67 in Rac and Cdc42) are rather significant

and probably contribute to the specificity of Rho proteins for ROCK, by affecting the overall

charge, hydrophobic and shape complementarity.

Comparison with other GTPase-effector structures. — Although the proposed RhoA contact

site I of PKN consists of the α1 helix (aa 25-28), β2 and β3 strands (aa 43-54) and the C-

terminal α5 helix (164-172) (Fig. 4), contact site II described as a symmetry related contact

site for the ACC finger (27), overlaps remarkably well with the ROCKI binding site described

in this study (Fig. 2D; Fig. 4). Except for four additional residues (N41, W58, S73 and D76),

PKN interacts with RhoA via the same amino acids as shown here for ROCKI-RBD (Fig. 2, B

and D; Fig. 4). Interestingly, the general type of interaction, i.e. the contact of complementary

hydrophobic patches stabilized at the edge by electrostatic interactions, is highly conserved.

The structure of ROCK-RBD is similar to the structure of PKN-RBD (27) in the respect that

they form a coiled-coil but the amino acids of PKN that contact RhoA at this site do not

correspond to those found in ROCK-RBD (Fig. 2, B and D; Fig. 4). This structural type of a

GTPase binding domain represents a new and clearly distinct category of target proteins in


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comparison with other known effector protein structures of the Rho family. The CRIB

(Cdc42/Rac interactive binding) motif of ACK (52), WASP (53) and PAK (54), for example,

is a largely extended structure containing a short region of anti-parallel β-strands. In contrast,

the Rac-effector p67phox contains four tetratrico-peptide repeats (38) and Arfaptin, which

consists of an elongated three-helix coiled-coil trimer, binds Rac1 in a nucleotide independent

manner (55).

Implications for Rho-mediated effector activation. — Effector activation by small GTPases

has been most extensively studied on PI3K, Raf kinase and Pak. For the Ras-PI3K interaction

it has been implicated that the recognition is provided by the interaction of the Ras binding

domain of PI3K with the switch I region of Ras whereas the activation is achieved by the

binding of the kinase domain to the switch II region (37). A similar model has been suggested

for the Raf kinase activation by Ras. The interaction between the switch I region of Ras and

the Ras binding domain of the Raf kinase may provide the initial binding process recruiting

Raf to the plasma membrane (56,57). This process is probably followed by the binding of the

cysteine rich domain (CRD) of the Raf kinase to the switch II region which may result in Raf

kinase activation (reviewed in 58). While the mechanism of Raf kinase activation by Ras is

still controversial, the Cdc42-PAK1 interaction has been suggested to induce a

conformational change resulting in the dissociation of the PAK1 dimer and subsequently in

autophosphorylation at several sites that prevent the kinase domain from reverting to the

inactive conformation (59-62). The structures of an autoinhibited α-PAK and the Cdc42·PAK

CRIB complex support this model (54,61).

The hydrophobic interaction of Cdc42 via L67 and L70 with PAK, ACK and WASP,

respectively, has been suggested to provide a common mechanism for activation of these

effectors (54). A similar mechanism can be hypothesized for the activation of Rho effectors


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since the corresponding leucines in RhoA, L69 and L72, are strongly involved in hydrophobic

interaction with ROCK (L1006, A1007 and M1010; Fig. 2 B and C) and PKN (L52, A56, L59,

V73 and L76; Fig. 2 D). The I1009A mutation has been previously defined as a dominant

negative mutant of ROCKI that abolishes ROCKI-RBD interaction with RhoA (13) and

inhibits Rho-induced formation of focal adhesions and stress fibers (63). I1009 is not directly

involved in the Rho-ROCK interaction but is adjacent to L1006 and M1010 (Fig. 2 A). Its

substitution may destabilize the Rho interacting coiled-coil motif and consequently the

complex formation with RhoA. The dominant negative effect of this mutant can be explained

by the following model: The ROCK I1009A mutant when overexpressed in cells sequesters

the endogenous ROCK (available at much lower concentration) by dimerization and the

formed heterodimer is apparently no longer activated by RhoA. It is important to note that

dimerization and autophosphorylation have been suggested to be major events for the

regulation of these kinases (34).

CONCLUSIONS

The presented structure highlights fundamental principles of small GTPase interaction with

effectors. The essential determinants of the GTP dependent interaction with effectors are the

two switch regions. Although nearly all effectors interact with switch I and switch II they

exhibit an amazing diversity in their structures. However, RhoA seems to interact preferably

with coiled-coiled domains. The structure described here represents a novel interaction type of

a small GTPase with a parallel α-helical coiled-coil dimer. We could show that the 160 kDa

protein kinase ROCKI employs only 10 residues of a 13 amino acid stretch at the C-terminal

part of the coiled-coil to bind a predominantly hydrophobic patch assembled by the switch

regions of RhoA. Although the presented structure of ROCKI and the recently published

structure of ROCKII alone do not form coiled-coils with the same twist, their C-terminal parts


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are structurally very similar. The specificity of ROCKI interaction with RhoA versus Rac1

and Cdc42 appears to be determined by the residues F39, Y66 and L69 of RhoA. They exhibit

a different orientation in RhoA concerning shape, charge and hydrophobic complementarity

with ROCKI. Our structure also suggests that the contact site between PKN-HR1a and RhoA

described as the symmetry related site in the structure of RhoA·GTPγS·PKN complex is most

likely the contact site II. However, important questions regarding the structural rearrangement

of full length ROCKI upon interaction of the RBD with RhoA and the implication of multiple

Rho binding sites in ROCK activation remain to be elucidated.


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Footnotes

The Abbreviations used are: GDP, guanosine 5´-diphosphate; GTP, guanosine 5´-

triphosphate; GppNHp, guanosine 5´-ß,γ-imidotriphosphate; HR, homology region; PKN,

protein kinase N; RBD, Rho binding domain; Sw I and II, switch regions I and II.

Acknowledgements. We thank A. Wittinghofer for continuous support and encouragements, S.

Narumiya for providing the cDNA of human p160ROCKI, Patricia Stege for expert technical

assistance, D. Fiegen, L.C. Haeusler and P. Chakrabarti for critical reading of the manuscript.

I. Schlichting and A. Scheidig for measurements at the beam line ID14-1 at the European

Synchrotron Radiation Facility (ESRF) and the ESRF staff.


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FIGURE LEGENDS

FIG. 1. Crystal structure of the RhoA-ROCKI-RBD complex. A, Schematic view of the

ROCKI domain architecture. The Rho binding domain (RBD, residues 950-1012) of ROCKI

is an integral part of the amphipathic α-helix; CRD, cysteine rich domain; PH, pleckstrin

homology domain. The Rho interacting motif at the C-terminus of the RBD (998-1010) is

depicted in red. B, The crystal packing diagram shows 4 molecules per asymmetric unit: a α-

helical coiled-coil of two ROCKI-RBDs (blue and cyan) and two RhoA molecules (gold and

beige). C, zoomed top view of the complex between two RhoA molecules (gold and beige)

and the Rho interacting motif of the RBD molecules (blue, cyan) shown in ribbon

representation. The bound GppNHp molecule (black) is shown as a ball-and-stick model.

Switch region I and II of both RhoA molecules are highlighted in red. The twofold symmetry

axis is depicted as a dot. D, Twist of the coiled-coil. The coloring of the ROCKI helices is the

same as in (B). The twist of the coiled-coil is specified above. Red arrows indicate the

location of ´stutters´. The box at the C-terminus shows the minimal Rho binding motif. E, The

structure of bovine ROCKII RBD coiled-coil (24) of which only the C-terminal part was

superimposed on the C-terminal part of the ROCKI RBD. The dashed-lined box points at the

Rho binding motif. F, Detailed comparison of the RhoA binding motifs of ROCKI (blue and

cyan) and ROCKII (light green and dark green). The depicted amino acids present the

residues of RhoA that contribute to binding of ROCKI.

FIG. 2. The RhoA-ROCKI-RBD interface. A, Stereo view of the final 2F0-Fc electron

density map of the minimal RhoA-binding motif of ROCKI-RBD (residues 998-1010).

ROCKI is shown in a coil representation colored with blue and cyan as in Fig 1B. Side chains

are shown as a ball-and-stick model. Electron density was calculated using CNS and

contoured at 1.5σ. B, Stereo view of the RhoA and ROCKI interacting residues. RhoA switch


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regions I and II (Sw I and Sw II) are shown in yellow, ROCKI is depicted as in (A). Residues

are labeled according to the standard numbering of RhoA and ROCKI and colored as in Fig.

1C. Electrostatic interactions are emphasized by dashed red lines connecting corresponding

atoms. C, Schematic representation of RhoA-ROCKI interacting residues. Dashed lines are

representative for hydrophobic (black) and electrostatic (red) interactions. D, Stereo view of

the symmetry-related interface between RhoA (yellow) and PKN (cyan). Residues are labeled

according to the standard numbering of RhoA and PKN.

FIG. 3 Determinants of the ROCK specificity towards Rho. A, Stereo view of

superimposed structures of RhoA (33) (orange), Rac1 (50) (yellow) and Cdc42 (51) (brown)

focusing on the ROCK binding region of RhoA. Amino acids are labeled according to the

standard numbering of RhoA. B, Surface representation of the GTPases in the same

orientation as in (A). The residues that interact with ROCKI RBD are colored as follows:

blue, positively charged nitrogen atoms; red, negatively charged oxygen atoms; green, non-

charged hydrophobic atoms; black, the oxygen atom of Y66. F39, found to be crucial for the

specificity towards ROCKI, is shown in yellow.

FIG 4 ROCK and PKN contact sites of RhoA. The contact site I proposed as the main

contact site of RhoA with PKN (27) is colored in brown. The common binding site of RhoA

for both ROCKI and PKN (contact site II) is highlighted in orange. The sites interacting

exclusively with PKN and ROCK are colored in yellow and red, respectively. All contact sites

were determined using the 4.5Å threshold.


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Table 1: Data collection, phasing and refinement statistics

X-ray data processing Resolution Observed reflections Independent reflections Rsym

a

Completeness Mean <I/σ(I)>

2.6 Å 43736 17511 6,4% (11.2%)b 95.9% (82.7%)b

24.0 (2.8)b

Molecular replacement statistics Resolution range Rotation (α,β,γ)c Translation (x,y,z)c Correlation coefficient Rcryst

e

15.0 Å -3.0 Å 138.30, 86.79, 279.71 319.48, 79.30, 271.05 49.85, -29.99, 47.11 -33.26, 77.19, 36.58 61.9 [45.9]d 39.2 [47.6]d

Refinement statistics Rcryst

e Rfree

e Mean B value R.m.s. bond lengths R.m.s. bond angles R.m.s. dihedral angles

21.9% 25.7% 37.0 Å2 0.007 Å 1.3˚ 21.9˚

a Rsym = 100 Σ|I-<I>|/ ΣI where I is the integrated intensity of a measured reflection. b In round brackets are quantities calculated in the highest resolution shell (2.69-2.60 Å) c Eulerian angles (α,β,γ) and orthogonal coordinates (x,y,z) for two RhoA molecules in the asymmetric unit. d In square brackets are quantities of the second-best solution. e Rcryst = 100 Σ|Fo-Fc|/ ΣFo where Fo and Fc are observed and calculated structure factor amplitudes. Rfree is Rcryst that was calculated using randomly chosen 10% of the data and omitted from the subsequent structure refinement.


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Dvorsky et al., figure 1


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Radovan Dvorsky, Lars Blumenstein, Ingrid R. Vetter and Mohammad R. AhmadianStructural insights into the interaction of ROCKI with the switch regions of RhoA

published online December 2, 2003J. Biol. Chem.

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structural insights into the interaction of rocki with the switch

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