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nature structural biology • volume 6 number 8 • august 1999 709

Copper is essential for life but also verydeadly if turned loose. Transition metalscan wreak havoc in cells via Fenton chem-istry, in which metals react with peroxideor superoxide to generate toxic hydroxylradicals. As a result, it has been necessaryfor Nature to devise an elaborate systemfor sequestering copper within the cell andfor delivering copper to various essentialenzymes such as superoxide dismutaseand cytochrome c oxidase1. The structuraldetails of how this occurs are coming tolight as a result of the work presented onpage 724 of this issue of Nature StructuralBiology2 and another recent paper inStructure3 from the same group. Thisstructural work follows closely on theheals in the biochemistry and molecularbiology of the copper trafficking system inyeast and humans where many of the newinsights have been made in only the lastfew years.

Overview of copper traffickingAt first, it would seem unnecessary to havea specific copper-insertion mechanism forenzymes like the Cu-Zn superoxide dis-mutase (SOD) since the affinity of SODfor copper is high — that is, in the rangeof 10-12 M. However, recent work by Raeet al.4 shows that the cellular concentra-tion of copper is on the order of 10-18 Mwhich amounts to less than one copperper cell. Such low concentrations of cop-per are maintained by proteins like metal-lothionein, together with a number ofchaperone proteins that directly deliverycopper to specific targets1.

Finding the correct home for copperbegins with a family of plasma membranecopper transporter proteins (CTR)(Fig. 1). One example is yeast CTR1, a46 kDa transmembrane protein5. The N-terminal region of CTR1 contains 11

repeats of a motif, Met-X-X-Met, which isalso found in prokaryotic proteinsinvolved in copper trafficking6,7. The pre-cise mechanism of copper uptake remainsunknown although the lack of ATPasemotifs suggest that ATP hydrolysis is notthe energy source8.

Once inside the cell, one fate of copperis binding to a metallochaperone (Fig. 1).One of the better understood of these pro-teins, Atx19, shuttles copper to a P-typeATPase10 called Ccc211. Ccc2 translocatescopper across the post-golgi vesicle mem-brane to the final target, Fet3, a copperoxidase required for iron uptake in yeast12.Atx1 appears to deliver its copper by directinteraction with Ccc29 which may involvea process whereby the copper is directlytransferred from the ligands of the donorto the ligands of the acceptor in a ligand-exchange mechanism. Moreover, anin vitro binding assay demonstrates thatAtx1 also readily exchanges bound mer-cury (Hg) with the metal-binding domainof Ccc23.

Structure of Atx1 The first step in unraveling the structuraldetails of metallochaperone functioncame with the X-ray structure of Atx1(Fig. 2; ref. 3). Solution of the structurewas not trivial in that the 1.02 Å Atx1structure represents one of the largest

solved so far by direct methods. Atx1belongs to a class of metallochaperoneswith the conserved Met-Thr/His-Cys-X-X-Cys metal binding motif. The structure(Fig. 2) shows, as expected fromsequence homologies, that the two Cysresidues in the conserved motif coordi-nate the metal ion. In the structure solvedby Rosenzweig et al.3, Hg occupies thesite, with Thr 14 close enough to the Hgfor direct binding interactions. Owing tothe conserved sequence surrounding theCys ligands, copper is expected to utilizethe same two Cys residues. The apo-Atx1structure is basically the same as the Hg-complex with the exception ofchanges in the metal-binding loop. Thisprecludes a mechanism for copper deliv-ery to the target protein involving largestructural changes resulting from metalbinding. The mechanism of target recog-nition remains unknown althoughRosenzweig et al.3 describe some possi-bilities based on the distribution of Lysresidues expected to be involved in form-ing an electrostatic interaction with the

Helping copper find a homeThomas L. Poulos

The crystal structure of the superoxide dismutase copper chaperone provides some key insights into themolecular mechanism of copper trafficking.

Fig. 1 Schematic representation of one possible model for copper transport. Copper first is trans-ported across the cell membrane using the CTR family of proteins. yCCS binds copper by anunknown process followed by formation of a yCCS–SOD heterodimer. Copper transfer from yCCSto SOD is followed by the assembly of SOD into active dimers.

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target. Recent mutagenic analyses usingan in vivo assay support this view13.

The copper site in Atx1 is quite differ-ent from many other copper-binding sitesthat are more fully sequestered within theprotein and have a more complete coordi-nation sphere. Obviously this is not desir-able for a metallochaperone, which mustbe able to release its metal to the correcttarget. One view holds that Atx1 forms anintermolecular complex with its targetwhich enables a simple ligand-exchangeprocess to occur in either a kinetically orthermodynamically favorable reaction9.The copper moves from the extracellularmilieu through the cytoplasm to its ulti-mate target via proteins that have pro-gressively higher affinity for copper orthat have rapid copper exchange rateswith their partners. For Ccc2 the finalstep requires energy in the form of ATPhydrolysis to translocate copper acrossthe post-golgi vesicle membrane, whichensures a unidirectional translocation ofcopper .

Structure of the SOD chaperoneA second major step forward in understanding copper trafficking, includ-ing some possibilities regarding chaper-one–target recognition, comes from theyeast copper chaperone for superoxidedismutase (yCCS) structure2. CCS deliv-ers copper to SOD through the formationof an intermolecular complex14 and isessential for conversion of apo-SOD to anactive enzyme15. Analysis of yCCS revealsthat CCS consists of at least two struc-turally well defined domains. The N-ter-minal domain is essentially the same asAtx1 except that no metal is bound and

the two Cys ligands form a disulfide bond.The larger domain is similar to SOD, aswas expected from sequence homolo-gies15, although the SOD-like domain ismissing the metal-binding sites. Thus, itappears that yCCS was pieced together byattaching a metal-free SOD-like domainto the C-terminus of the Atx1-like domainthrough a flexible linker containing a Gly-Ala-Gly sequence.

It was known from biochemical studiesthat human CCS can interact directly withSOD14 and the yCCS structure providessome intriguing ideas about how thismight occur. yCCS crystallized with twomolecules in the asymmetric unit and theyCCS dimer interface is strikingly similarto the SOD dimer interface, suggestingthat yCCS–SOD recognition may involveformation of a yCCS–SOD heterodimer(Fig. 3). Examination of the yCCS andSOD dimer interfaces reveals striking sim-ilarities. A model of the hypotheticalyCCS–SOD heterodimer generated bysuperimposing yCCS onto SOD revealsthat the heterodimer interfaces may beremarkably similar to the SOD or yCCSinterfaces. Note, however, that the metalsite in the yCCS Atx1-like domain is ~40 Åfrom the metal sites in SOD (Fig. 3).Therefore, in order for direct coppertransfer to occur, there must be substan-tial motion of the Atx1-like domain sothat it is in a position to donate copper to

710 nature structural biology • volume 6 number 8 • august 1999

the SOD-like domain. This appears feasi-ble since the two domains are held togeth-er by a flexible linker and the domaininterface is not very extensive.

As intriguing as this idea appears, recentwork by Schmidt et al.16 has demonstrat-ed, using an in vivo assay, that yCCS lack-ing the Atx1-like domain still leads tocopper insertion into SOD and that theAtx1-like domain appears to be importantonly under conditions of limiting copper.Furthermore, these same authors haveshown that Cys 229 and Cys 231 in the C-terminal region of yCCS are essentialfor activity16. This suggests that these twoCys residues coordinate the copper that isultimately delivered to SOD.Unfortunately, the last 26–27 amino acids(223/224–249), including Cys 229 and231, are not visible in the yCCS crystalstructure. However, if we assume that theyCCS–SOD heterodimer is basically thesame as the SOD dimer, then the last visi-ble residue in the yCCS structure,Glu 223, would be 26 Å from the SODmetal sites. There is a sufficient number ofamino acids between Glu 223 and the Cysligands, residues 229 and 231, to bridgethis 26 Å gap and place the Cys residueswithin contact distance (~4 Å) of the SODmetal sites. In this scenario the ‘disor-dered’ C-terminal region binds copperwhich can either facilitate direct bindingto the SOD metal sites for direct ligandexchange or simply increase the effectiveconcentration of copper near the SODmetal sites.

Certain problems and questions arisewith this view. For example, what roledoes the Atx1-like domain play in yCCS ifit is not essential for copper delivery toSOD? Perhaps this domain simply servesto recruit copper under copper-limitingconditions16. More troubling from thestructural perspective is the requirement

Fig. 2 The crystal structure of Atx1 complexedwith mercury3. Atx1 is a small 74-residue pro-tein whose structure was solved by direct meth-ods. The overall fold of Atx1 is similar to that offerredoxins. Cys 15 and 18 provide 2.33 Å and2.34 Å, respectively, linear bonds to Hg whileThr 14 is 3.05 Å from Hg for a weaker interac-tion. The coordinates utilized for generatingFigs 2 and 3 were generously provided byA. Rosenzweig and were prepared with theprogram SETOR17.

Fig. 3 This is a view of theyCCS dimer highlighting theexpected location of the SODmetal ions (yellow balls)assuming that the yCCS–SODheterodimer is basically thesame as the yCCS and SODdimers. Note that the Atx1-like metal site is quite far(>40 Å) from the SOD metals.The location of the last visibleresidue in the yCCS structure isindicated as C-term. Thisresidue, Glu 223, is closeenough to the SOD metal sitessuch that an extended sectionof polypeptide could place theCys 229 and Cys 231 copperligands within contact dis-tance of the SOD metal sites.

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of forming a yCCS–SOD heterodimer,considering that this would necessitatedisruption of the quite stable SOD dimer.Is it possible that yCCS forms a het-erodimer with newly translated SOD, thus‘capturing’, if only transiently, an SODmonomer? Despite this fly-in-the-oint-ment about the stability of the SOD dimer,the striking similarity between the yCCSand SOD dimer interfaces makes forma-tion of a heterodimer as the basis forrecognition a rather attractive hypothesis.As with most novel and unexpected struc-tural results, the current yCCS structureand its implications with respect to metal-lochaperone function will undoubtedlyreceive intense scrutiny and testing.

AcknowledgmentsI would like to thank T. O’Halloran and V. Culotta for

nature structural biology • volume 6 number 8 • august 1999 711

valuable discussions and access to ref. 16 prior topublication, and A. Rosenzweig for atomiccoordinates.

Thomas Poulos is in the Departments ofMolecular Biology & Biochemistry andPhysiology & Biophysics, and the Programin Macromolecular Structure, Universityof California at Irvine, Irvine, California926197-3900, USA. Correspondenceshould be addressed to T. P. email:poulo@suci.edu

1. Valentine, J.S. & Gralla, E.B. Science 278, 817–818 (1997).2. Lamb, A.L., Wernimont, A.K., Pufahl, R.A.,

O’Halloran, T.V. & Rosenzweig, A.C. Nature Struct.Biol. 6, 724–729 (1999).

3. Rosenzweig, A.C., Huffman, D.L., Hou, M.Y.,Wernimont, A.K., Pufahl, R.A. & O’Halloran, T.V.Structure 7, 605–617 (1999).

4. Rae, T.D., Schmidt, P.J., Pufahl, R.A., Culotta, V.C. &O’Halloran, T.V. Science 284, 805–808 (1999).

5. Dancis, A., Yuan, D.S., Haile, D., Askwith, C., Elde,D., Moehle, C., Kaplan, J. & Klausner, R.D. Cell 76,

393–402 (1994).6. Cha, J.S. & Cooksey, D.A. Proc. Natl. Acad. Sci. USA

88, 8915–8919 (1991).7. Odermatt, A., Suter, H., Krapf, R. & Solioz, M. J. Biol.

Chem. 268, 12775–12779 (1993).8. Dancis, A., Haile, D., Yuan, D.S. & Klausner, R.D. J.

Biol. Chem. 269, 25660–25667 (1994).9. Pufahl, R.A., Singer, C.P., Peariso, K.L., Lin, S.,

Schmidt, P.J., Fahrni, C.J., Culotta, V.C. & Penner-Hahn, J.E. Science 278, 853–856 (1997).

10. Moller, J.V., Juul, B. & le Maire, M. Biochim. Biophys.Acta 1286, 1–51 (1996).

11. Yuan, D.S., Stearman, R., Dancis, A., Dunn, T.,Beeler, T. & Klausner, R.D. Proc. Natl. Acad. Sci. USA92, 2632–2636 (1995).

12. Askwith, C., Eide, D., Van Ho, A., Bernard, P.S., Li, L.,Davis-Kaplan, S., Sipe, D.M. & Kaplan, J. Cell 76,403–410 (1994).

13. Portnoy, M.E., Rosenzweig, A.C., Rae, T., Huffman,D.L., O’Halloran, T.V. & Culotta, V.C. J. Biol. Chem.274, 15041–15045 (1999).

14. Casareno, R.L., Waggoner, D. & Gitlin, J.D. J. Biol.Chem. 273, 23625–23628 (1998).

15. Culotta, V.C., Klomp, L.W., Strain, J., Casareno, R.L.,Krems, B. & D., G.J. J. Biol. Chem. 272, 23469–23472(1997).

16. Schmidt, P.J., Rae, T.D., Pufhal, R.A., Hamma, T.,Strain, J., O’Halloran, T.V. & Culotta, V.C. J. Biol.Chem., in the press (1999).

17. Evans, S.V. J. Mol. Graphics 11, 134–138 (1993).

Src autoinhibition: let us count the waysStevan R. Hubbard

Src family tyrosine kinases are key cellular signaling enzymes whose catalytic activities are tightly controlled.Recent structural and mutational studies have revealed additional intricacies in the autoinhibitorymechanisms by which catalytic activity is repressed.

The Src family of non-receptor tyrosinekinases comprises nine members includ-ing Src, Blk, Fgr, Fyn, Hck, Lck, Lyn, Yesand Yrk. These signaling proteins cat-alyze the transfer of the g phosphate ofATP to specific Tyr residues in proteinsubstrates, leading to the modulation ofprotein–protein interactions or enzy-matic activities. When activated, the Srcfamily tyrosine kinases play key roles inthe control of cell proliferation and dif-ferentiation, and therefore their catalyticactivities are subject to tight regulation.Early biochemical studies suggested thatthe various domains within Src werecritical for keeping its catalytic activityin check1,2. The landmark crystal struc-tures of Src3,4 and Hck5 published twoyears ago provided vivid snapshots of thedomain interactions that serve to represstyrosine kinase activity. The recent pub-lication of crystal structures of Src6 andHck7 in Molecular Cell, and a mutagene-sis study of Src reported on page 760 inthis issue of Nature Structural Biology8,

provide new insights into the molecularmechanisms of Src autoinhibition.

Cooperative domain interactionsThe domain organization of Src and itsfamily members consists of a myristoy-lated N-terminus, followed by a non-conserved region, a Src homology 3(SH3) domain, an SH2 domain, a tyro-sine kinase domain (also known as anSH1 domain), and a short C-terminaltail (Fig. 1a). The modular SH2 and SH3domains, which are also present in a hostof other signaling proteins, bind to spe-cific sequences in proteins that containphosphotyrosine and polyproline,respectively9,10. Src family kinases con-tain two important sites of tyrosinephosphorylation with opposing regula-tory effects11. Autophosphorylation ofTyr 416 (numbering is for Src) located inthe activation loop/segment of thekinase domain leads to the stimulationof kinase activity. By contrast, whenTyr 527 within the C-terminal tail is

phosphorylated by a non-receptor tyro-sine kinase, Csk, repression of Src kinaseactivity occurs12. Previous biochemicalstudies strongly suggested that this nega-tive regulation was due to the intramole-cular binding of the SH2 domain of Srcto its own Tyr-phosphorylated tail13. TheSH3 domain was also shown to influencethe catalytic activity14, but the molecularmechanism by which this occurred wasnot well understood.

The original Src3,4 and Hck5 crystalstructures provided striking verificationof the interaction of the SH2 domain(blue) with phosphorylated Tyr 527(orange) (Fig. 1b). Yet the most dramaticand unanticipated finding that emergedfrom the crystal structures was the inter-action of the SH3 domain with the linkersegment between the SH2 domain andthe kinase domain. While several Srcfamily members, such as Hck and Lck,contain within their SH2-kinase linkerregion a Pro-X-X-Pro motif — the mini-mum consensus sequence for SH3 bind-

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