`J\SSOCIATION FOR Tl !H
`· AnvANCEMENT or
`SCIENCE
`
`•
`CIE
`
`CE ISSN Q036·8075
`
`10 NL\\' 1991
`VOLUME 2)2
`NU.Ml\Ell 5007
`
`7S5 This \Veck in Sde11.-c
`
`7S7 Technology f<>r A1nerira's Future
`
`763 Science in the Persian Gulf: ·r. l\1. BuYcE 11 i\hnh Problen1s: T. lvL i\1lJRl'HY;
`l-.t \V. LEVJ~E; R. 1). l-IANSON; C. B. I·IATPJELn; D. E. KosHLAND, )ll. n Energy
`Savings: A. R. l.O\'INS; Ci. ~1. RAILN\\'ELL M EPA Con1n1ittcc: M. E. O'CONNOR•
`Cryst,1] Structure of Bi·c-Venn1n Phospholipasc A2 : Corn·C'tion: 11. L. ScoTr,
`Z. Onv1NOW.SKI, f\1. I J. GELB, P.H. SIGLER Ill i\-ktnbr;.\tlC·Bound Phosphotyrosinc
`Phosph~1L1sc~: A. J'. Vv'll.LIAMS
`
`767 S\\•eeping O\'erhcad r.1tcs under th<' n1g; go11nbling \\'ith Poker Fh1t sdC'ncc; etc.
`
`lt1ltin1orc Thro\VS in the TO\\'cl u !),1vid Raltin1orc's 1\1ca Culpa
`768
`771 The Tn1e Source of HIV~
`772 Science Under \\'r;tps in Prince \Villiam Sound
`773 Science Ac.tdc1ny Elects Ne\V Nkn1bers
`774 Rrlefi11gs: Hidden Costs of the Sp.1cc Station a A Big Gift fro1n Big Oil • A Billion
`Bucks tOr 1\-l;1tcrials Ill Congressional I>ay t1 Ten Years for the Brain a Cuban All).S
`Control D Biotechnolot~Y Exct·s Earn l\1orc 11 Correction
`
`776 Engineering Dognrn. Gives VVay to Chaos ii Plying High v1ith Ch:1os Control
`778 A Ne\v Hall Cian1e in Nuclear Physics
`779 Ho\\' Peptide Honnones Get Ready for \Vork
`781 Praying: ~·ianris:es Play 'l'op Gun
`782 Sex and the Single Gene
`783 Deep Rocks Stir the 1\1a11tk Pot
`
`789 Reproductive Behavior and I lc;llth in Consanguineou~ f\1arri.1gcs: J\. 11. llrrrLES,
`\V. M. MASON, J. GREENE, N. A, K,\O
`795 Neutron Sc1ucrin~: Progre.~s and Prospect~; J, f). AXE
`802 Divcrsily of(J Proh:ins in Sign<ll T1ansdw .. ·tion: 1\·1. I. SIMON, 1\1. P, STR/\THMANN,
`N. GAurAM
`
`809 Zinc Fingcr-11NA Recognition: Cr>'st.11 Stnicttirc of a Zif268-DNA Co1npkx- at
`2.1 A: N. P. PAVLETICH and C. 0. PABO
`817 A Ne\V Cofactor in a Prokaryotic Enzy1nc: Tryptophan T1yptophylquinone as the
`Rcdox Prosthetic Group in i\·icthyl:uninc l)chydroge1rnsc: W. ~. f\1clNTIRE,
`D. E. \'VEMMEtl, A. C1usro.sE1tuov, M. E. L1usntOM
`
`II
`
`II
`
`SCIENCE (ISSN 003.6·8075) ls publ\•hcd w~okly on fr!day, o~cept the IBsl v.aek Jn OecembH, by Iha American
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`S<::.,oce 'n l1'lmeir1 L!<YJ10!<5
`
`7.'>2
`
`SCIENCE, VOL. 252
`
`
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Our results show that the presence of the
`fcmak-spedfit: tM and the tra-2 product~
`pro1notc fcn1alc·spedfic splicing of dsx pre(cid:173)
`rnRNA and tlrnt the 13-nt sequences in the
`fc1nalc exon and the fcn1alc·spccific acceptor
`sequence participate in regul~tion of dsx
`cxprcs.~ion. The IM and tr.r-2 product.~ m~1y
`interact directly 'vith the 13-nt sequences in
`the female·spedfie cxon, and such interac(cid:173)
`tion,<; may allo\v the suboptimal female-spe(cid:173)
`cific polypyrin1idinc stretch to be recognized
`as a splicing signal) thus resulting in cn(cid:173)
`lrnnccmcnt of the wa; of the fr1nalc-~pcl:ilic
`acceptor site.
`
`REFERENCES ANf> NOTES
`
`I. R, Nothigu and M. Steinmann-Zwick}', Trmds
`Genet. l, 209 (19SS); T. \V. Clin~, in Ori,i:/lr 1md
`f;'p,,forfon o_{S.~\·, H. 0. Hah'Ofson and A. Monroy,
`Ed;. (l.i>-s, Nnv Ymk, 1985), pp. 301-327; 11. S.
`B1ker, N.11mt 340, 521 (1989).
`2. B. S. Ihker Jnd K. A. llldg<", Grn<lits 94, 383
`(19fif)).
`3. R. S. fbka rnd M. F. \\'olfnn, (;,.,,,., f),·r" 2, 477
`( 1988); K C. llurti1 ~ra:l fl.. S. ll:iker, C:rll !i6, 997
`(1989).
`4. R. N. N~goihi, M. McKcown, K. C. l\11rti>, J.M.
`Bdrn<.\ R. S. B.tkcr, C..·l/ 53, 229 (J\.lSii).
`5. M. l>kKcuwn, J. M. fidute, R. T. Boggs, Ibid., p.
`887; B. A, Somowski, J.M. Ildotc, .\t ~kK<.-own,
`ibid. 58, 449 (1989).
`6. K. Inoue, K. Hn1hijinn, H. S.1bmmn, Y. Shi111111~,
`N.111u~ 3·14, 461 {19?0).
`7. T. &huplud1, f)r!'. Ri«oJ. 1\9, 117 (l?l\2); J. M.
`Bcloie mrl R. .~. Ral..<"r, 1'1,-.r. N~tl. Ar,,t/, Sri. ll.S.11.
`79, l.'i61\ (1982)> net•. l!i,1/. 95, .'il2 (19R::\).
`ll. H. Amrein, M. Curm;in, R. Norhign, Cd/ 55,
`1025 (19RR); T. J. GorJ.ldci, J.-F.. F.d<trom, Jl. S.
`R.tkcr, ibitl. 56, 1011 (19R9).
`9. C. C. Qutl)', ll. C. lknde)', J. D. Keen<', rbid, 57, 89
`(1989); I. \'I. Man1j, ibid., p. L
`10. J. M. Uc~, ~i. McKc"Own. R. T.
`ll.
`lluAA,>,
`Ohkl1H, Il. A Sornow>!<i, Dc1,. Geiu1. IO, 143
`(1989).
`I I. ·nu-, 1cgio11 from at1<i including the entire thir.t C'l'.OI\
`to the P\il II site loc.i.tcd 1284 bp dowr.1tecm1 of the
`frmak ac~eptor site, and the ugioo from the Ace I
`~ite lonied 250 bp upHreJ.m of the mite acceptor
`site to the site lo;;;,1ted 6+ bp dO\\H>ttt-Jm ol th<
`donor .1itc' of th<' 6fth <'.lon, were j.oinni .i.ntl in.1crted
`intn the copia vector.
`12. ~L MrKrown, J.M. Belote, R. T. Bogg~, Cd/ 53,
`81\7 (1988).
`I.~. K. Ho;hijimt d ~/., unpubfohcd ob1crYJtiorn.
`14. R. N. Nagruhi .1.nd Il. S. l!Acr, Gr!!<> V<i•. 4, M9
`(1990).
`15. The ,J~y f1agment thll ntcnd.1 from the third cxon to
`1128 hp <kiwrntre.un of thr frmtk .Kc<ptor 1it~ and
`whid1 exclude> the po!y~dcuyhtion ~lgnal (Af) was
`fw,ed to theji"' frngmcnc that <onuins .l. portion of
`tho.: i1111ou and tl1( fol101\·i11g ;.c.:oml nnt1 {lurn:·
`1pon,iing (0 bl1o!5 825 w 2534 11f the pul.J!hl1cd
`IC<JUCrKC [A. J.~ughr>il :md Ill. I'. Sw!I, NnlWt' 310,
`2$ (1984)]}. The- 1cmlti11g fogmem Wl' itbn!cd
`into th<: copi~ \'Cdor.
`16. T~ rcgk"">O Jlom the thin( cxon (138 bp) to the site
`48 hp do1'n>tT<"'.llll of the donor site of the third
`nuu, md the rq;ion from the >itc 250 bp 11p11rern1
`(>f the nHlc .Kccpt"r ~itc w the ,,ir;,, 64 hp down·
`strcJm uf dx· du11ur ~ite ur t.hr fifth c:mn, W<:re
`joincJ <U:d irnc·rt<Xl irnu th<> rnpil 1·e,t<.>r.
`17. ·111<" copil·<h\ ddetiun mutanl~ wer<: rnmtruned :i..<;
`fo!luw;:
`the regiom between 345 to •160 bp
`(Rl•5·6), 234 to 459 bp (R5·6), 190 RI 480 hp
`(R6), 34-4 to 599 bp (Rl), ot 234 to 599 bp (RO)
`Juwmtre.IJ11 uf the fr nu.I~ acceptor site were dele1ed
`from copil·{k\, .ut<l Kpn I liuhr> \\nC inse1ted. A
`pnl~111er.i<<: ch\in raaion (I'C[l) frlt;incnt that
`(ontair.l"ti the r~gion 268 to 627 hp downstrc-.un of
`the frnuk·.spc.:i!i<' ancpcor ,<ire wa• i1c>ertc<l ;lt the
`Kpn l linker of the ltO cumtruct in 1he corrcn
`
`836
`
`orientation (SR) or in 1he opposite oritm~tion
`(ASR). PCR w,\1 perfonnt"d e55enti.i.lly 011k-<nibed
`[R, K. Sliki a .11., &fro«' 239, 487 (1988)].
`18. 'M. R. Gr>:cn,.-hwu. R.v. Uoul. 20, 671 (191:!6).
`19. OligomKloo!kk·dirccted muwgene:>is 1n.1 per·
`fom1cd c,.;cnti.1!1r o.s Jc.scribed !~L /. Zolkr ~nti M.
`Smith, ,\fahrJs En.;;ym>'I, 100, 468 (1983)].
`20. 'lhc r'giou 34U w 1128 bp duwmtrc11u ut' tht
`frm1lc J.;ccptor .site w<i.i dcktOO from copi~·dt7.,
`
`cripil-.lt7.(Pyll\), and coph·dt1.(Py13). rre·mRNA
`exprc55ed from the dektion COMtrna nf rnpil·dtz
`w.i~ spliced at the ji.z ac~~ptor .'iitc n'l'f! in tlli:
`prc"-!ncc of the lr<f and lfJ-2 prnduct< in Kc cc\ls.
`21. \VethmkM.McKoown forf1.idJNA~11<l B. Amrein
`f°' tiu-2 cDNA. SuppottN hr Gr.l.Ilt-in-Ai,l
`621l6St109 for 1~dllly pmmoiOO rt"-!..'Mch frnm 1])(
`l\linLHry ofFdu.:atio.1, ~ci~llCe and Culture of Japm.
`1 NOl·,·m\o:r 1990; ,1cc~ptc<l l MMch 1991
`
`Solution Sttucture ofFKBP, a Rotamase Enzyme and
`Receptor for FK506 and Rapamycin
`STEPHEN w. MICHNICK, MICHAEL K. ROSEN, THOMAS J. WANOLF.SS,
`MARTIN I<ARrr.us 1 * STUART L. Scr-nlETI1ER *
`
`Inm1tu1ophilins, ,vhen eo1nplexed to inununo~upprcssivc ligands, appear to inhibit
`signal tr<lnsduetion patlnv;lys that result in cxocytosis and transcription, 'The solution
`structure of one of these, the hun1an FI(506 and tap.unycin binding protein (FKBP)>
`has been dctern1ined by nude<lr 1nagnetic resonance (NMR). PK.BP h;ts a previously
`unobserved antiparalh:I l~-1:1hcct folding topology that results in <l no\•el loop ct·ossing
`and produces a large cavity Lined b)' a conserved array of aron1atic residues; this cavity
`serves as the rotaniase active site ;ind drug-binding pocket. 'fhere arc other .~igniticant
`structural f'c<llure.~ (such as a protn1ding positively charged loop and an apparently
`flexible loop) th;tt nrny be involved in the biological activity ofFJ(UP.
`
`F KRP TS ,\ $OUiHLE, C'ITOSOLTC. 1u-:(cid:173)
`
`ceptor ( J, 2) for the iln1nunon1pprcs·
`sanrn FK50fi and rapa1nycin (3). Both
`_
`FKBP ;u1d cydophilin (4--6), which is a
`recepttir fi>r the immun11~u11pre.<;.~ant cyclo·
`sporin A (CsA), cat•llyzc the interconversion
`of cis- and trans·rotan1ers of the pcptidy!(cid:173)
`prolrl an1idc bond of peptide and protein
`sub.~tr:ltes. These rot;m1ase.~ ouc inhibited by
`their respective immunosupprcssivc ligands.
`f..1.l"Chanistic studies suggest that a complex of
`i111nu1nophili11 (in1nnu1osupprcss.uu binding
`protein) and lig.u1d interferes v;ith the ir1n-.1cd(cid:173)
`lul.u· tra.nspott of proteins in\•olvcd in sign:tl
`tran~dnction p.uhways <l~socfated \Vith hath
`exocytosis (7) and trouiscription (J> 8).
`In this report \\'e presen[ the solution
`structure of hun1an FKRP obtained by nu·
`clear Overhauser eflCct (NOE)-rcstraincd
`n1olecular dyo•ln1ics (r1'1D) simulations (9).
`The NlviR stnKtures satisfying the NOE
`11nd empirical energy function ro:straints
`have backbone root-1ne.u1·squarc deviations
`(RlvfSDs) from the relined averngc structure
`in the range of 0.80 to 1.4 A for tht' f.
`strnnds, 0.19 to 0.40 A for till' ct bdb:, and
`1.02 A to 1.72 A for all residues except 83
`to 90 (sec below). i\iany side d1ain~, partic(cid:173)
`ularly aro1n,ltics, are 'vell defined. FKBP has
`a novel fiilding topology in .that two lnops
`that co1uicct the str,lnds of an ;u1tiparallcl ~
`sheet cross one another. Hydrophobic side
`
`Dtpanm.:nt of Chcmi1t1y, H.irvlt<l llnivn!ity, C1m·
`bridge, MA 02.138.
`
`'To whom rn1re1pondenct st10uld bl' 'u:klrt<-.~d.
`
`chains in the core of the protl'in form a
`fargc, deep pocket that includes the rota-
`1nase acti\•e site and the drug-binding site.
`The structure of the binding site appears to
`be high!}' conserved in related FK506- and
`r.1pamycin-binding proteins.
`Sequence-specific assign1ncnts of 92% of
`the obscrv.1b[e 1H resonances in FKBP have
`been n1adc by use of a co1nbination of
`hou1onuclcar and hcteronude.tr two-tlimen·
`sinn;il NJ\-IR rechnique~<> as reported earlier
`(10), Stn1ctur<ll restraints for the dyn.unic5
`siinulations were obtained through empiri(cid:173)
`cal c;11ibr.ltion of cross-peak magnih1de.s in
`nuclear O\'crhauser effect
`spectroscopy
`(NOESY) spectra recorded with 1nixing tin1es
`
`1 07
`
`Fig. 1. a·C~rhon riblXJn drawing of hmn.in
`IlKBP; th<' li\•c·~tr,,nded [l sheet, C( helix, and
`connecting !oops arc indicated, a~ wd! ;1$
`the
`COOl-1· and NHi·lccmini and <.:c:rtain K>idm:~ of
`interest.
`
`sr:JHNCE, VOL. 2SZ
`
`
`
`/
`
`10 t-.1AY 1991
`
`Fig. 2. (A) The u·carbon tr:ux~ uf 13 >trucnHfS
`gcneraled by n:~tiaintd molecular dynamics are
`~hown in blu(', (SA)<<h which includes side chaim
`of aromatic residues in the drug· binding pocket,
`is ~hown ;1S the bcJ.V}' line. Aromatic side chains
`:in: colnr·rodcd: Trp, yellow; Pbe, orange; and
`Tyr, grccu. The NHi· amt COOH-ccunini ;ire
`irul.ic1t.::d witli green and red dot ~urfaces, u:~pcc
`tivcl)'. (13) (SA)icri showing ]ocition of the ligand
`binding site formed hy rhc ft sheet .md then hdix;
`certain re5iduc~ arc numbered. (C) Exp.u1dcd
`view of (SA).,r, showing crossing of the !oops
`connecting the lirst and IU11rrh (rekl'ant residul!!;
`in orange) and the fifth aud M:um<l (rdcv.1nt
`rc.~iducs in yellow) str-J.nds of the ~sheet; km>wn
`interstrand hydrogen hond~ arc indk.ltrd. Rele(cid:173)
`vant residues in tht: third strand nc shown in
`green. (0) Hydrophobic residties in the drug(cid:173)
`biuding pt.X:kt:t of (SA},.,p Side chains ;ire color(cid:173)
`coded ;l.5 in (A), with 01ddi1io11J[ ;1fiphatic rc~idurs
`in purpk.
`
`of 50, 100, 150, and 200 ins versus known
`distances in regular second;i.ry stnKlural cl(cid:173)
`e1nents (11). Of particular use in una1nbig(cid:173)
`uouslr dett:nnining long-range NOEs W{'rc
`t\vo FKBP sampks biosyuthetku.ll}' dcuter(cid:173)
`ated at the methyl protons of Leu and Ile
`and of Val and lie (12, 13). A total of 860
`NOE-derived distance constraints \Vas used
`in the simulations. In addition, 4~ Xi dihe(cid:173)
`dral angle restraints (in 1hc range ±60° or
`± 12<f) !lnd 44 backbone q, angle rc.st1aints (in
`the r.1ngc ±60°, ±SOa, or :±-40') were used; the
`angle con'itraint<> are ba.'ied on analrsi<; of cou(cid:173)
`pling constants and cros.s-pcak nugnilucle.s
`1nc;\sun:d in correlated sp<:<.troscopy (COSY)
`5petLrn, and on NOE dat,1 ( 1'1). Jn 32 cases, X;
`could Ix n.--suictc<l to a ±60° r.mgc, allowing
`stcrcospecific a._--signtncnt of side chain protons
`h:i~ed on in!r;wcsidue NOE~ front NI-I and
`CaH. Hydrogen bond constraints \Vere u~ed
`for 25 slo,vlr exchanging :un!dc protons. Sev(cid:173)
`enty-nine N()Es, six hydrogen bonds, and six
`backbone dihedral angles \VCrc dctcrn1ined in
`an iterative n1anncr bast:d on unrefined, re(cid:173)
`strained StLucturcs. The ri\1D sinndations
`\Vere carried out by using a si1nulatcd anneal(cid:173)
`ing protocol \Vith the prograsn X·PLOR (9).
`Statistics indicative of the accuracy and preci(cid:173)
`sion of the cxpcri1nenta!lj' detern1ined stn1c(cid:173)
`n1rc of FKRP and a de.~cription of the proto-
`
`R.EPORTS
`
`837
`
`
`
`l'OI used ro gener,uc these srrucn1res are
`provided in ·rabk 1.
`The solution stn1cturc of FKBP (Pigs. 1
`and 2) is ch.1ractcrizcd br a large an1-
`phiphilk, antiparal!rl fivc-.~trnndcd ~ sheet
`with +3, +l, -3 1 +l tupulogr (15). Thc
`str;\nds of the sheet arc cotnposed of resi(cid:173)
`dues 1 to 7, 20 to 29, 46 to 49, 71 to 771
`and 96 to 107. An :unphiphilk n: helix,
`fonned
`to 65, packs
`fron1 rc.'iiduc.~ 59
`against the hydrophobic face of the sheet at
`an angle of 60~ 'vith n•spcct to the long axis.
`The helix is tethered to the fifth and si:\'.ond
`.<1tr.u1ds of the sheet by nine- and fi\•c-rcsiduc
`loops, ri:spcctivcly. The sheet h;ls a right(cid:173)
`handed twist ;u1d \\'r.1ps around the helix to
`fonn a 'veil-ordered hydrophobic core (Fig.
`l); this structure is in accord \\'ith :l large
`n111nher of long-r,u1ge ~idc chain N()F. in(cid:173)
`lt-rauiuns. Bad~bont' hydrogen-bond pru(cid:173)
`pcnsitic.\ arc satisfied !Or the ~ slwct by
`intcrstr,uui a1nidc-carhon)'I conrncts and !Or the
`a helix by intcrrcsiduc a1nidc-c.ubonyl i, i+3
`conracts. 'l11ere are no side chain hydrogen
`bonds bt:t"'een the helix and the sheet, indic.1t(cid:173)
`ing st.1hiliz.uion of tht'!iC ~tnKhircs thron~\h
`hydrophobic (van dcr Waal<;) interactions. The
`loops connecting the~ Stl",\11ds cont.1in sccond(cid:173)
`•UY sn11cturn1 clcmcnt'I (indnding scvcrnl ~
`tunt'i} that satisfy soinc of the backbont' hydro·
`gen bond propt:n'>itie.'i, 'll1c loops arc \Yell
`ddim:d by 1nediu1n- and !ung-nu1ge Nl)E cun·
`tacts t'Xcept for t\\'O rcgiot1~, rrsichies 37 to 43
`and 83 to 90 (sec l~ig. 2, A .u1d B).
`A not.lblc fc,1tur.: of l'KBP resulting frotn
`the +3, + l, -3, + l topo!ogyofthc p sheet is
`a topological cros'>ing of the loops Ser8-Gly19
`and Lcu 50-Gln70
`• A view of this region of the
`protein is shown i11 Fig. 2C. Although crossing
`top-0!ogics ha\'c lx-en observed in protein~ ron·
`t:iining paralli.:I f~ sheer,.; ( /6), the}' "'ere pre·
`stunrd to br forbidden in antiparallcl sheets
`(17). The absence or loop crossings ha.~ been
`;'ttributcd to the dillicultic.'i of obt,Uning dfi(cid:173)
`cient side chain p<irking, of satisfying the hy(cid:173)
`drogen bond p1ope1t~itics of the backbone
`amide.~ of both segments, .tnd of dc8cribing a
`~iinpk !Olding pathw,ly (JS). In FKBP, the
`structure of the cro"-'iing ~cgnicnts is deter-
`1nirn:d by NC)E~ induding '11u 14-&r67
`, G!r11
`-
`&r67, Arg 13-Scr<'7, Prol<'-V:tl68, and Pro 16-
`Leul0'•. There are van dcr \.Yaals t:ontacts bc(cid:173)
`t,vecn the side chain_'i of Prot6 mul J.eu 11)(',
`Va!0
`1-l and Leuw 3 , :tnd Thr 14 and \ 1111611
`, The
`hydrogcn-OOnding p1opcn.~itil·.s uf nll-111)' resi(cid:173)
`in the Cto.\~ing region (Pro9, Asp11 ,
`due.>
`1
`111r1
`, and Val68
`, lvktM', Scr67
`) an: smisficd
`·
`through backbonc-b.Kkbone and b,\ckbone(cid:173)
`si<lc chain interactions. All ;u-nidc protons in
`the inner strand and lwo a1nidc proto11~ in the
`outt'r str;\rnl (I,ys 17 01n<l Gln20
`) cxcholngc slo\\'·
`ly when the prott:in is di~oln::d in DJO; this
`result i~ indkati\'e of hydrogen bonds stabiliz(cid:173)
`ing this topology.
`
`. ~·
`
`T
`!
`
`In a pre!i111inary sn1dy of the FKBP(cid:173)
`FK506 and FKBP·rap;ln-1ycin co1np!cxes we
`noted NO Es fron1 the indole ring of 'l'rp59
`and several other \1nidcntifi.:d aron1,uic pro·
`tons to the pipicolinyl ring of the hound
`drugs (/J). Bai.t:d on the solution con!hrn1a(cid:173)
`tion of FKBP, it 'vas possible to dl'li11c the
`binding region as an cxtcnsi\'C aron10Hic dus(cid:173)
`, Phc46
`ter cotnposcd of 'l'yr26
`, Phe-1 8
`,
`, Tyr82
`, and Phc9
`Trp59
`'1. The.~c residues
`pack together with a number of aliph~1tic
`residues to tOrn1 a large, well-defined hydro·
`phobic core (Fig. 2D). 'l'hus, FK506 and
`rapainycin bind in the hydrophobic pocket
`that consists of a twisted and curved anti par(cid:173)
`allel µ-sheet platfonn with \'l,\lls fonncd by
`an a helix :tnd a long loop (Fig. 2B). The
`loc.,tion and detailed structure of the bind(cid:173)
`ing .~ite h,1:; been contirn1ed by the .~uhsc
`qul·nt x-ray strw:tlire uf the FKBP-FK506
`con1pkx (19}. The ;1rchitcct11rc of thl~ bind(cid:173)
`ing site, \Yhosc naturnl ligands art' not
`
`k.no\\'ll but n1ay include peptides, is very
`difiCrent fron1 thos.: fanned by 13 barrels in a
`nun1ber of protein~ that bind hydrophobic
`ligands lsuch as 1'2 1nyclin protein (20) ;1nd
`bilin binding protein (21)]. Ho\VC\'er, al(cid:173)
`though thc overall structures an: very difiCr(cid:173)
`cnt, the FKBI' binding site has clcn1e11ts in
`com1non with that of the class I tnajnr
`histoco1np.ltibility con1pkx (i\1HC) glyco(cid:173)
`prorcin HLA-A2 (22) 1 \vhich has a flatter
`;111tip;1r;1\ld p-shcet platfonn \Vith
`\Valls
`forrned by nvo !ong r'I helices. Peptide $Cg(cid:173)
`ments that frJrn1 loops at the open end of the
`pocket, spccilicaHy, J\spJ 7
`to Asn-l.i and
`G!y82 to Ile90
`, arc not well dclincd (see Fig.
`2, A and B). The i.Kk of !ong·rangc NOE.<;
`in both ~eg1nent5 coupled with prc\·iou:o.
`cvidc11ce of conforlll<Hiona] excl1augc in the
`second ( 10) indic1tc th;tt lhf'.y rnay be tkxi(cid:173)
`blt: in ~olutiun. A Ctnnp;irison of 11-1 and
`lf,N cl1c1nic;tl shifts obt,1incd from single·
`qlrnnnlln coherence (SQC) ~pcctra of L5N-
`
`Table 1 •. ~tntctur.11 and energetic statiHic.~ fi:ir FKfll'. Structure dctem1ination \\'.lS ;;chicnxl with;\
`simulated ,1nncaling prot()(ol consisting of thtcc stage~: (i) a conformational ~e.trch phase; (ii) an
`annealing phaw; and (iii) a refit11:rncnt pha~c (9, 30), Nurnbns in par~·ndu:~cs arc the number of
`restraint~ in each class or the number of bonds, angles, and dihedrals in tht stn1ct1.1re geometry
`st~tistin. SA; is the ith structure oht'1incd hy the protocol given ahnvc. 'r11c S1\ cnlmnn gi\'CS the
`avccJgc and stand:trd dcdations for the indkatcd varilbks obt,1incd from the 21 ~tructure..\ with the
`!ownc empirical cncrgil'~ ;1nd rc~idul! viubtion~ of cxpcrimtnta! rntraint.;, (SA),.r reprc>cnts tht
`a\·erag1.· ~tn1cture of SA1 !east·sqnJre fit to each other including all atonu (e:.:n:pt for residues 83 tu
`90) and rcfinl."d with 1000 .~tcps ofstccpc.\t"dc.1ccnt energy minimization. The R.\1SDs arc from the
`\lpper or lower bountl~ of the distJ.Jtcc rcstr.1int.s; none of the strucrnres ~howctl dc\'i:ltion$ gre.ltcr
`1han 0.25 A. ReMraints were da.-,sificd into the following cJtcgocie~ ba.\t'tl 011 :-trong, nitdium, :rnd
`wc.1k NOE~ (J 1): backbone, 2.5 :I: 0.5 A, 2.75 -± 0.75 A, 3.S l: l.S A; side clui11, 2.5 l: 0.5 A,
`3.0 ± 1.0 A, 3.S ± 1.5 A; 0.5 A wa~ added to the upper limiB for diManccs invoking methyl
`protons. Aver.ige lli\fSDs and stand,trd deviation~ of the !lnJ! srrucmtcs SA, ag.1inst the relined
`average ~tn1crun:: (SA),d· For P ~beet: b.1ckbone, l.01 ±' 0.16; ;ill ;1toins, 1.92 ± 0.14. Fur a hdix:
`b1ckhonc, 0,31 ± 0.07; all atonu, 1.05 ± 0.16. For all atoms CXC('pt residue~ 83 to 90: lnckbone,
`1.45 ± 0,20j all aton\5 1 2.49 ± 0.17.
`
`Parameter
`
`RAfSDs_Ji-cm1 r.\·11··rimi-11t11/ dl#,111re r.-ilflli1111 (AJ
`0.024 ±
`(910)
`
`(246)
`(7.fi4)
`(39)
`(146)
`
`I 121
`(SJ
`(2')
`(133)
`(50)
`
`0.011 ~t
`0.1124 ±
`0.0·1 l ±
`().031 ±
`
`0.008 ±
`0.012 ±
`0.018 ::!:
`0.023 ±
`0.034 ±
`
`0.002
`
`o.oo:'l
`0,003
`0,008
`0.004
`
`0.010
`O.OIR
`0.008
`U.004
`o.oos
`
`0.008
`
`0.022
`0.00R
`0.021
`0.051
`
`0.024
`0.003
`0.000
`n.020
`o.n.17
`
`Tot,11
`Peptide backbone rcstuint~
`l111r.ircsidue
`lntcrrc~iduc .~equcnti:il (11 jl = 1)
`Interresiduc short-r.u1gc (li-j1 ~ S)
`Intcrcc~itluc luug-r;iuge (li--:/1 > 5)
`Sid('-d1,1in restrJints
`lntr.ue;;iduc
`Intcrrcsidue bcquenthtl {Ii-JI = l)
`lntcrrc~idw: ~horr-1.1ng;e {Ii-JI :,; S)
`lntecrc:-idue lon!J;·r.1ngc (li-jl > 5)
`Hrdrogcn·b(md restr,1inu;'
`Deviations from idcali7_cd gcomctn.---
`Bumb (A)
`'
`Angks (degrees)
`Impropcrs (degrees)
`
`E,,~>I
`li1.x,_,J
`E•nr.k
`E,1·1c.<1.,1
`
`(1682)
`0.013 ±
`3.360 ;I;
`(3047)
`0.460 ±
`(498)
`p,1/t·miiil 01ci:i:J' slalistlcs (J.mi//111,1/)
`_!. 120
`-2280.0
`-2170.0
`±
`49.9
`3.1
`·17.8
`± 20
`652.0
`486.0
`±
`532.0
`512.0
`IR
`15.8
`16.S
`l.8
`±
`EitnpcH;"•
`±
`-336.0
`IS
`.~58.0
`R,-J"
`± 120
`-3200.0
`-2920.0
`E<latramtio
`5.9
`1.7
`5.6
`:t
`Bc1,w~
`±
`22.3
`27.0
`3.9
`ENnR
`'Backbone hydrogen ll<lnd IL-Straint.< were ;15sig11e<I to rJ . .ngcs 'NH 0 = 1.9 ± 0.5 A rnd '"'-u "" 2.8 :!: 0.5
`A.
`tidcJh7.td g<'O!nrofrs bJse1l on CHAR,\lM p.lr~m~tas (JI).
`
`0.000
`0.064
`0,030
`
`O.Ol3
`3.240
`0.490
`
`SCll!NCE, VOL. 25£
`
`
`
`r ,.
`' ,,
`
`I
`
`bbckd FKUP and the 15N-hbeled FKBP(cid:173)
`FK506 co1nplex suggests that significant
`d1;lnges occur in rhe.<;e loop regions follo,v(cid:173)
`ing drug binding. Thus, the distinct inhibi(cid:173)
`tory effect:> that result frorn PK506 and
`rap.l!n)'cin binding (R) rni'.)' i1n1olve rhcir
`ittlluencc on the gco1ncff)' of thc~c loops.
`\Ve have recently provided evidence sug(cid:173)
`gesting th•H the n1cchanism of roramase
`catalysis is due 10 no1n-ovalenr .~tabiliz.ation
`of a tv/1ste<l :unide in the transition state of
`the re-action (2J, 24), rather tlrnn fonnation
`of ;l covak-nt tctrahcdr<ll intennediatc. A
`sin1ll.1r 1ncchanis1n has been proposed for
`rhe rornn1ase cydophilin (25, 26). All Ly.~,
`Ser, and Thr side chains in FKBP arc direct(cid:173)
`ed away from the active site, and the Ca of
`Cys22 is 11 A fron1 that ofTrp59 to\vard the
`narr<>\V end of the n1okcule. The pipccolinyl
`ring ofFKS06, \Vhich contacts Trp59
`, prob(cid:173)
`abl}' 1niinics the proline ring of a natural
`peptide substrate that is subject to rotaniase
`cataly ... is. ·n1us, the aforcn1cntioned rc ... idues
`<lrc too far fron1 the active site to add to the
`peptidyl-prolyl <lmi<le carbonyl and facili(cid:173)
`t<'te rotation about the C-N bond. Sitc(cid:173)
`<lirccted nn1tagenesis is being used to dcter(cid:173)
`n1inc the role of the tyrosines and other key
`residues found in the active site (Fig. 2D).
`Sever.1! highe1· rnolecular \\'eight PK506-
`<ln<l rap:unycin-binding prot1..·ins have re(cid:173)
`cently been reported (27). The in11nunophil(cid:173)
`ins of 1nolccubr weight 13,000 and 27,000
`cont.lin FKBP-likc do1nains of~ 110 an1ino
`acids that .share high .sequence identity to
`FKBP (28). Arornatic residue~ that corre(cid:173)
`1 'I'yr82, and Phe"9, 'vhich
`spond to l'rp"9
`line the dn1g-hindi11g pocket, :1re conserved in
`thc..;,e proteins and in <111 FKBPs identified fm1n
`different orgimisn\.'i to date (8), s11gge.~ting tlrnt
`the ligand-binding pocket is si1nilar in all llK(cid:173)
`BPs. Thus, the prc~cnt stnKlure of lnunan
`FKBP 01.\y be rckv.1nt to undtrstanding not
`only its o\Vn cnzr1natk and dnig-binding
`properties, hue those of all 1ncn1lx:n; of this
`trncrging fan1ily of proteins.
`J\loft• 11ddctl in Jlff•f!f A report h;ts ;1ppc.1rcd
`noting the identity of PK.BP to inhibitor-2 of
`protein kinase C (PKCI-2) (29); howc~'cr, we
`find that 1:1mp docs not inhibit the kinase
`activity of isolated protein kinase C or protein
`kinase C-n1ediated events in cell.,. (32).
`
`REFERENCES Ai"'ID NOTK<;
`
`l, .\f. \\'. H:lrding etlll., N.u11re 341, 758 (1989).
`2-. J. J. Skkicrkl et <1/., ilid., p. 755.
`3. B. E. llicr<:r <I~/., Pr • ..:. •'-'"II . ..-k~J. S<i. U.S .• ·!. 87,
`9231 (1990).
`·L R. E. lfa.udschuml(hu ct.;/., Sckt:u 226, 544 (1981).
`5. G. Jlhh(r rt al., .'V"t111e 337, •i76 (1989).
`6. N. T~blnsl1i, T. H.ay.uin, M Sumb, i~,·d., r 473.
`7. T. Hu!tsd1 rt,;/., P1.:f. N,,//. AN./. Sri. C:, S. A., i11
`\)lN.
`R ,<:, J .. .Srhrctha, Scimu 251, 18.~ (1991).
`9. /\. T. l\rirnga~nd M. Karpll.1>,Au. Cliu11. Rf-<. 21, 54
`( l'.l'Jl) for n:vir.w; R. Kapkin, ll. H. I'. Zu:,krwcg, H.
`:\\.Scheel:.., R. floclem, \Y. F. \'.1U Gtuist~nn,}. kf,>I.
`
`lOMt\Y 1991
`
`B!M. 182, 179 (1985); Ii. T. Briinger, G.M. C!oce,A.
`M. Gro1xnl"V£11, 1{. K.urlm, Pt<'<. Nari. rLd. Sd,
`U,SA. 83, 3801 (1986); J\f. Nilg~. G. M. Clnii:, A.
`M. C.roumlll•u1, FEBS I_,·11. 229, 317 (19.'IR); M.
`Nil~, in Co111p!•l.lliN1.1f A<J\onrcfti''lt.- Sr11dy ~(Bid'>,.>k<JI
`,\forr,111;.•.'u-"&.< bJ' !\',\II~, f. C:. Hcxh, l'.d. (Pbl\!m,
`New Yori-., iu pr~<.~); A. T. Briingn,X-f'/.0R .\f~ru11f,
`Va.<irn 2.1, (Y.1k Univcr>it)', '.'·kw fl~1;:n 1990)
`Ill. M. K. Rmcn, S. W. Midu1td, M. K.npl11,, ,'\. I.
`Schrdher, Iliahm1iJlfJ', in prcs.s.
`11. t\. Km1ur, n. W.>gl\Cr, R. ll. l'm<.t, K. Wiithri...i1, .f
`/lr•1. C1ic111. SN, 103, 36S4 (1981); M. I', \\'illi.1Jn't>n
`dwl.,j. Al,•l. llid. 182, 295 (1985),
`12. R. F. Sumhen, A G1hr, G. L. Verdin.:, S. l..
`Schrd~<, NMmr: 346, 671 ( 1990).
`13. T. J. 'Vandle<s, S. \V. ,\1ichnkk, M. K. Rusen, M.
`Karplus, S, L. Schreiber, j. Ar~. C/11:111. S,v, 113,
`2339 (1991).
`14. A. Pardi, .M:. Billeter, K. \\'Uthrich, ]. hfol. Biol.
`180, 741 (1984); D, Neuhaus, G. \VJi,'ller, M.
`\'auk, J, H. R. Kagi, K. \Vihhrid1, R111,.f. Dir.</1tm.
`151, 257 (1985); G. \'IJ.gner •I ,11.,}. hr~/. Hi"/.
`196, 611 (1987); E. R. P, Zuidenng, R. Ilodcm,
`R. Klptein, Blor<'lymus 24, 601 (1985)<
`lfi. J. S. Rich.i.r(hon, f'-.!,1/1m: 268, 495 (1977),
`16. For e1;:1mple, gyu.--ml<lchp.le phruph.ue d~h)'<irogem<e
`(D. Mora.1 ti~/., j. llfol. (J;e,n. 250, 9137 (1975)],
`17. C. Chuthi.1.,,-Jn1111. R.;11, Bi,xf1cm. 53, 537 (1984);
`Y, N. OiirgJ.dze,Aa.i Cry1llfll~~r. i\43, 405 (1987J.
`18. 0. B. l'ti~yu anJ A. V, Fi11kd~tci11, Q. R.:11. Bfo(cid:173)
`ph}'s. 13, 339 (1980).
`19. G.D. V.1.n Du)n<', R. JI. StJnd.\ctt, I'. A. Kup!us, S.
`L. Schrdb~r, J. Cbrd}', &/f11re, 252, 839 (1991).
`20, T.A. Jones, T, llcrgfors, J, Scdzik, T. Voge, R\IUO
`]. 7, 1597 (1988).
`21. R. Huber cl .ii.,]. ,\fol. RiN. 198, 499 (1987),
`22, I'. J. lljml:.niant/ "'·· Natiu<' 329, 506 (l9R7); J/,id.,
`p. Sl2..
`23. M. K. Rmen, R. 11. St~nd.Krt, A. CJ.lit, M. N.ikat(cid:173)
`,uk-.., S. I.. Schreib<r, Sciwu 248, 863 (1990)
`24, M. \\'.Albers, C. T. \V-ath, S. L. &·hreilxr,]. Org.
`Chrn1. SS, 4984 (1990).
`25. J. Liu, M. Altx.1:-, C. Chen, S. I.. Sdueilxr, C. T.
`\Va.lsh, f>rN. Narl.Ar,,J. Sd. /J,S./\. R7, 2.!H-1 (1990).
`26. R. K. Harri>on and R. L. Stein, Bfr-llmrmtry 29,
`l6S4 (1990).
`27. II. Fn:tz.,·1.,1.,]. Am. CJ;e111. s,,,_ tl3, 1409 (l'JYI).
`28. Y.-/. Jin, l>L \\'. Allxrs, R. P.. Ilierer, S. I.. Schreiber,
`S. J, Rumkolf, Prcv. NrTtl . .'lr,;J. Sd. U. S. A., in
`pre~. A. G~l~t, \V. S. Llllc, lt. f. Stambcit, S. L.
`Schrd~r, in prcp.irJtion.
`29. bl. G. Goeb!, Cd! 64, 1051 (1991).
`30. Surling Hl\tctllrts for the simuh1cJ .lnn<:aling pro·
`to..-nl H'>fl>i1tcd of FK.lll' polyrcpti1lc ch.1ins with
`rwdnni h.tckh<mc dihedn! ~nglc":S. Tht conformJ·
`ti um.I 1<:-Ard1 phnt u.imbtt·d of 20 p> uf 1110lccl1!Jr
`
`dynamic~ .lt lOIJU K umkr the: influence of~ fora
`fid<l Ccdsc-..I for simuluc,j ann,~!ing ~tudie$, A
`quartk 1.:p11hion pntcntial wa.1111cJ in thi: f<>r<<~ &lrl
`i11 phu: uf the ~tamiird CHARMM ''.lll dcr \''31]~
`po[<'nthl for purpu~.1 of coinput.itionll dlicicncy.
`The fut(c ~um!rnl o!" the n:pu!>i>.C pokntiil (liltl!·
`l'EL) WJS snkd w 0.002 kcJI 11101- 1 A- 1 during1hc
`confomutional ;carch to .1Jlow .itum.1 w piv; ticdy
`tluo1igh c.ld1 utha. 'l11t. 11i111ncc re>tr a int potemial
`tMget fnmtion (E:-;01;) w.•~ a HjUJJc wcU with
`luunonic wa!h Jt the uppc·r aml !uw.:r hound> ofrhe
`Ji>urxc rc<crlinu and J lurrnonic phi1 linelr .1wi1(h·
`ing ftl!\(tion a1 0.5 A .di.we the upper hound. The
`h.1u1l0nic force corn.mm WJ.> SO kcal mo]··l A 2 and
`the Jincir component Jud a ~lope uf 5 kc-JI mot- 1
`A- 1
`• The purpo;e of thi1 phl...::: W.'~ 10 ohuin an
`unbiJ.SOO search of confo1m~tional space with 3 force
`fiekl doininAtixl Uy the npedmcutal nrgel function.
`The annc.1\ing: ph.1..1c corui1tcti of two .'!Jg<i. First,
`dl1dng o lO·p> intervll tlie slope of th~ linear com(cid:173)
`ponent uf lf:-.:o£ w.i.s itKr>:-J.~cd tu 50 h~l mu)-1 A - 1
`~n<l the scak of L'rulrEI, w.1s r.ai.!cd to U.l kcal mot- I
`A- 1. Second, the di1tJ1Ke r<"'o'itrJ.int pounti1l wa.<i
`nude hlrmonk .J.t b-Oth the lower and upixr dist.1nce
`bounds, opcrim<ntal rlihed1.1J f(Stuints w.:re intro(cid:173)
`du(ed, !he scale ofE1wi'HL W.1.S incre,\\td to 4.0 kc.al
`moi·-l A .. \ a.nd the tcm~ntmc wu adj1111cd from
`1000 K to 300 Kin ~teps uf25 K orer 2.8 ps. The
`npciimcntJ/ dihedral potential (H<-1t.J WJ> J squ~rc
`wdl \1ith !urmonk walk Jt the uppu ind lowa
`bounds with a fon::~ constlnt of 200 k.<:al mo1- 1
`r~d-· 1. The rdincmcnr pha.1ecomi<rcdof 1000 st(p;
`nf sta~>t de:><::ent minimil.Jtion in which the full
`CHi\!lMJ\t force field induding tk.:-tro~~tic an•l
`V.lll o.kr \I/HJ. nonbomlcd ll"llll> l\"<."1e U~td (JI).
`Sra1i1tk.sw(rc.:ak:ubted with the progrJ.JTI X-Pl.OR
`(9). Energy a1uJy5is w.J.S perfom1a.i wi1h th<: ~l~ll
`dard CHA.MfM force field (JI).
`~I. II. It. ll1<X1h rl ~/.,}. (;1m1rot. Cilni1. 4, 187 ( 1983).
`32. /\!:. \V. Alb::rs a ,1/., llfo111td. C/iu11. Lw., in pf(ss.
`33. Coordin1t~ oftl\.! 21 firu..l sm1ctun--;; am! nfth<: mini(cid:173)
`rniu....J
`a1·~-i3gi: 'lru<.t1uc ·will bt: d<.cf-x.;ir.xl
`in the
`HrooHnwn Prouin Dat.t Hmk along with ,l lilt of lil
`npcrii1ie11tJ.] re>\rJ.i11u. . .'>upp.)rtcJ by the N~tiL>nJ.l ln(cid:173)
`,\frdiol .<>.ima~ (GM-;'18627,
`,1rimrr. of rlcrv.:ol
`nw.mkJ w S.L.S; GM-30804, a11·m~e.! tu M.K.).
`N11km.;.l S<:ieucc FcUJ1illtiui1 pre<lvctur~l fcllO\nhip; tu
`M.K.H. Jnd T.J.\V. ~oc gr.1.tdi1Uy xkoow!.ulj;o.:I. W<:
`th.i.rlk S. Slumb.i)'Jti foe pnintiog out the now!ty of the
`ioa1> lRt:..-,,ing tup.1!ogy iu FKllP. NM.It ~p:<..trJ \\<:r<!
`obtJln.:<l thrnugh the Jmpirc> of the I hrv.i.rd Uniwr·
`~ity Dqn1tmmt ofChcnilltry Irt1lnunror~tHm l.cntcr,
`which w.>1 supported by Nill grmt l-SIO·RR01870
`3nd NSF gr mt CHE 88-14019.
`
`29 Janwry 1991; lcc-eptetl l