S1

Small Molecule Gated Split-Tyrosine Phosphatases and Orthogonal Split-Tyrosine Kinases

Karla Camacho-Soto, Javier Castillo-Montoya, Blake Tye, Luca O. Ogunleye, and Indraneel Ghosh*

Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona, 85721

ghosh@email.arizona.edu

Supporting Information: Figure S1. Protein tyrosine phosphatase alignment. (p. S2-S7) Table S1 and Sequences. List of constructs for loop insertions and split-proteins designed and used in this study. (p. S8-S17) Table S2. List of constructs with respective vectors (p.S18) Figure S2. Effect of rapamycin addition on lysate and phosphatase splits activity. (p. S19) Figure S3. Small molecule dependent luciferase reassembly with the PYLcs/ABIcs

*//ABA and GID1/GAI//GA3 based CID systems. (p. S20) Figure S4. The activity of the FKBP/FRB//Rapamycin dependent split-Lyn fragments at three different dissection sites. (p. S21) Supplementary Methods (p. 22)

S2

Supplementary Figure S1. Alignment of Protein Tyrosine Phosphatases (PTPs).1,2 The boxed areas in gray for PTPRA, the first PTP in the alignment, corresponds to areas considered for loop insertions.

PTPRA/hPTPalpha|P18433|241-501 ------FREEFNALPA-------------C--PIQATCEAASKEENKEKNRYVNILPYDH PTPRB/hPTPbeta|P23467|1703-1963 ------LSKEYEELKDVG---------------RNQSCDIALLPENRGKNRYNNILPYDA PTPRC/hCD45|P08575|651-910 ------FLAEFQSIPRVF------S---------KFPIKEARKPFNQNKNRYVDILPYDY PTPRD/hPTPdelta|P23468|1357-1612 ------FSQEYESIDP-------------G--Q-QFTWEHSNLEVNKPKNRYANVIAYDH PTPRE/hPTPepsilon|P23469|135-394 ------FREEFNSLPS-------------G--HIQGTFELANKEENREKNRYPNILPNDH PTPRF/hLAR|P10586|1352-1607 ------FSQEYESIDP-------------G--Q-QFTWENSNLEVNKPKNRYANVIAYDH PTPRG/hPTPgamma|P23470|848-1119 -------------FSEDF------EEVQRCTADMNITAEHSNHPENKHKNRYINILAYDH PTPRH/hSAP1|Q9HD43|820-1079 ------FADKYQQLSLVG---------------HSQSQMVASASENNAKNRYRNVLPYDW PTPRJ/hDEP1|Q12913|1041-1298 ------FAEEYEDLKLVG---------------ISQPKYAAELAENRGKNRYNNVLPYDI PTPRK/hPTPkappa|Q15262|887-1141 ------FKEEYESFFE-------------G--Q-SASWDVAKKDQNRAKNRYGNIIAYDH PTPRM/hPTPmu|P28827|900-1154 ------FKEEYESFFE-------------G--Q-SAPWDSAKKDENRMKNRYGNIIAYDH PTPRN/hIA2|Q16849|709-969 ------LAKEWQALCAYQ--------------AEPNTCATAQGEGNIKKNRHPDFLPYDH PTPRN2/hIA2beta|Q92932|745-1005 ------LEKEWEALCAYQ--------------AEPNSSFVAQREENVPKNRSLAVLTYDH PTPRO/hGLEPP1|Q16827|938-1195 ------FSLQFEELKLIG---------------LDIPHFAADLPLNRCKNRYTNILPYDF PTPRQ/hPTPS31|Q9UMZ3|2036-2292 ------FQEEFSELPKFL---------------QDLSSTDADLPWNRAKNRFPNIKPYNN PTPRR/hPCPTP1|Q15256|393-647 ------LQSEFMEIPMNF-----------------VDPKEIDIPRHGTKNRYKTILPNPL PTPRS/hPTPsigma|Q13332|1393-1648 ------LSQEYESIDP-------------G--Q-QFTWEHSNLEVNKPKNRYANVIAYDH PTPRT/hPTPrho|O14522|889-1143 ------FKEEYEALPE-------------G--Q-TASWDTAKEDENRNKNRYGNIISYDH PTPRU/hPTPlambda|Q92729|888-1144 TAEGYGFKQEYESFFE-------------G-------WDATKKKDKVKGSRQEPMPAYDR PTPRZ/hPTPzeta|P23471|1717-1992 ------FTEEFETLKEFY------QEVQSCTVDLGITADSSNHPDNKHKNRYINIVAYDH PTPN1/hPTP1B|P18031|3-277 ------MEKEFEQIDKSGSWAAIYQDIRHE--ASDFPCRVAKLPKNKNRNRYRDVSPFDH PTPN2/hTCPTP|P17706|5-275 ------IEREFEELDTQRRWQPLYLEIRNE--SHDYPHRVAKFPENRNRNRYRDVSPYDH PTPN3/hPTPH1|P26045|646-901 ------VLIQFEQ--LYR-----------K--KPGLAITFAKLPQNLDKNRYKDVLPYDT PTPN4/hMEG1|P29074|655-911 ------VLTQFDQ--LYR-----------K--KPGMTMSCAKLPQNISKNRYRDISPYDA PTPN5/hSTEP|P54829|300-555 ------LQAEFFEIPMNF-----------------VDPKEYDIPGLVRKNRYKTILPNPH PTPN6/hSHP1|P29350|244-515 ------FWEEFESLQKQE-----------V--KNLHQRLEGQRPENKGKNRYKNILPFDH PTPN7/hHePTP|P35236|97-350 ------LEEEFLKIPSNF-----------------VSPEDLDIPGHASKDRYKTILPNPQ PTPN9/hMEG2|P43378|303-574 ------IYEEYEDIRR-------------E--NPVGTFHCSMSPGNLEKNRYGDVPCLDQ PTPN11/hSHP2|Q06124|247-521 ------FWEEFETLQQQE-----------C--KLLYSRKEGQRQENKNKNRYKNILPFDH PTPN12/hPEST|Q05209|28-293 ------FARDFMRLRRLS------TKY---RTEKIYPTATGEKEENVKKNRYKDILPFDH PTPN13/hPTPBAS|Q12923|2213-2467 --------PSKELENLQE-----------L--KPLDQCLIGQTKENRRKNRYKNILPYDA PTPN14/hPTPD2|Q15678|909-1180 ------VFTEYEQIPKK------------K--A-NGIFSTAALPENAERSRIREVVPYEE PTPN18/hBDP1|Q99952|26-291 ------LAGEFSDIQACS------AAW---KADGVCSTVAGSRPENVRKNRYKDVLPYDQ PTPN21/hPTPD1|Q16825|896-1167 ------VFTEYERILKKR-----------L--V-DGECSTARLPENAERNRFQDVLPYDD PTPN22/hLyPTP|Q9Y2R2|24-289 ------FANEFLKLKRQS------TKY---KADKTYPTTVAEKPKNIKKNRYKDILPYDY PTPN23/hHDPTP|Q9H3S7|770-1130 ----DTVWRELQDAQE----------------HDARGRSIAIARCYSLKNRHQDVMPYDS

S3

PTPRA/hPTPalpha|P18433|241-501 SRVHLTPVEGV--PDSDYINASFINGY-------QE-KNKFIAAQGPKEETVNDFWRMIW PTPRB/hPTPbeta|P23467|1703-1963 TRVKLSNVDDD--PCSDYINASYIPGN-------NF-RREYIVTQGPLPGTKDDFWKMVW PTPRC/hCD45|P08575|651-910 NRVELSEINGD--AGSNYINASYIDGF-------KE-PRKYIAAQGPRDETVDDFWRMIW PTPRD/hPTPdelta|P23468|1357-1612 SRVLLSAIEGI--PGSDYVNANYIDGY-------RK-QNAYIATQGSLPETFGDFWRMIW PTPRE/hPTPepsilon|P23469|135-394 SRVILSQLDGI--PCSDYINASYIDGY-------KE-KNKFIAAQGPKQETVNDFWRMVW PTPRF/hLAR|P10586|1352-1607 SRVILTSIDGV--PGSDYINANYIDGY-------RK-QNAYIATQGPLPETMGDFWRMVW PTPRG/hPTPgamma|P23470|848-1119 SRVKLRPLPGKDSKHSDYINANYVDGY-------NK-AKAYIATQGPLKSTFEDFWRMIW PTPRH/hSAP1|Q9HD43|820-1079 SRVPLKPIHEE--PGSDYINASFMPGL-------WS-PQEFIATQGPLPQTVGDFWRLVW PTPRJ/hDEP1|Q12913|1041-1298 SRVKLSV-QTH--STDDYINANYMPGY-------HS-KKDFIATQGPLPNTLKDFWRMVW PTPRK/hPTPkappa|Q15262|887-1141 SRVILQPVEDD--PSSDYINANYIDGY-------QR-PSHYIATQGPVHETVYDFWRMIW PTPRM/hPTPmu|P28827|900-1154 SRVRLQTIEGD--TNSDYINGNYIDGY-------HR-PNHYIATQGPMQETIYDFWRMVW PTPRN/hIA2|Q16849|709-969 ARIKLKVESSP--SRSDYINASPIIEH-------DPRMPAYIATQGPLSHTIADFWQMVW PTPRN2/hIA2beta|Q92932|745-1005 SRVLLKAENSH--SHSDYINASPIMDH-------DPRNPAYIATQGPLPATVADFWQMVW PTPRO/hGLEPP1|Q16827|938-1195 SRVRLVSMNEE--EGADYINANYIPGY-------NS-PQEYIATQGPLPETRNDFWKMVL PTPRQ/hPTPS31|Q9UMZ3|2036-2292 NRVKLIADASV--PGSDYINASYISGY-------LC-PNEFIATQGPLPGTVGDFWRMVW PTPRR/hPCPTP1|Q15256|393-647 SRVCLRPKNVT-DSLSTYINANYIRGY-------SGKEKAFIATQGPMINTVDDFWQMVW PTPRS/hPTPsigma|Q13332|1393-1648 SRVILQPIEGI--MGSDYINANYVDGY-------RC-QNAYIATQGPLPETFGDFWRMVW PTPRT/hPTPrho|O14522|889-1143 SRVRLLVLDGD--PHSDYINANYIDGY-------HR-PRHYIATQGPMQETVKDFWRMIW PTPRU/hPTPlambda|Q92729|888-1144 HRVKLHPMLGD--PNADYINANYIDGY-------HR-SNHFIATQGPKPEMVYDFWRMVW PTPRZ/hPTPzeta|P23471|1717-1992 SRVKLAQLAEKDGKLTDYINANYVDGY-------NR-PKAYIAAQGPLKSTAEDFWRMIW PTPN1/hPTP1B|P18031|3-277 SRIKLHQE------DNDYINASLIKME-------EA-QRSYILTQGPLPNTCGHFWEMVW PTPN2/hTCPTP|P17706|5-275 SRVKLQNA------ENDYINASLVDIE-------EA-QRSYILTQGPLPNTCCHFWLMVW PTPN3/hPTPH1|P26045|646-901 TRVLLQGN-------EDYINASYVNMEIPA----ANLVNKYIATQGPLPHTCAQFWQVVW PTPN4/hMEG1|P29074|655-911 TRVILKGN-------EDYINANYINMEIPS----SSIINQYIACQGPLPHTCTDFWQMTW PTPN5/hSTEP|P54829|300-555 SRVCLTSPDPD-DPLSSYINANYIRGY-------GGEEKVYIATQGPIVSTVADFWRMVW PTPN6/hSHP1|P29350|244-515 SRVILQGRDSN-IPGSDYINANYIKNQLLG---PDENAKTYIASQGCLEATVNDFWQMAW PTPN7/hHePTP|P35236|97-350 SRVCLGRAQSQ--EDGDYINANYIRGY-------DGKEKVYIATQGPMPNTVSDFWEMVW PTPN9/hMEG2|P43378|303-574 TRVKLTKRSGH--TQTDYINASFMDGY-------KQ-KNAYIGTQGPLENTYRDFWLMVW PTPN11/hSHP2|Q06124|247-521 TRVVLHDGDPN-EPVSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVF PTPN12/hPEST|Q05209|28-293 SRVKLTLKTPS--QDSDYINANFIKGV-------YG-PKAYVATQGPLANTVIDFWRMIW PTPN13/hPTPBAS|Q12923|2213-2467 TRVPLGDE-------GGYINASFIKIP------VGKEEFVYIACQGPLPTTVGDFWQMIW PTPN14/hPTPD2|Q15678|909-1180 NRVELIPTKEN---NTGYINASHIKVV------VGGAEWHYIATQGPLPHTCHDFWQMVW PTPN18/hBDP1|Q99952|26-291 TRVILSLLQEE--GHSDYINGNFIRGV-------DG-SLAYIATQGPLPHTLLDFWRLVW PTPN21/hPTPD1|Q16825|896-1167 VRVELVPTKEN---NTGYINASHIKVS------VSGIEWDYIATQGPLQNTCQDFWQMVW PTPN22/hLyPTP|Q9Y2R2|24-289 SRVELSLITSD--EDSSYINANFIKGV-------YG-PKAYIATQGPLSTTLLDFWRMIW PTPN23/hHDPTP|Q9H3S7|770-1130 NRVVLRSG------KDDYINASCVEGL-------SPYCPPLVATQAPLPGTAADFWLMVH

S4

PTPRA/hPTPalpha|P18433|241-501 EQNTATIVMVTNLKERKECKCAQYWPD--QGC--WTYG--NIRVS--VEDV-TVLVDYTV PTPRB/hPTPbeta|P23467|1703-1963 EQNVHNIVMVTQCVEKGRVKCDHYWPADQDSL---YYG--DLILQ--MLSE-SVLPEWTI PTPRC/hCD45|P08575|651-910 EQKATVIVMVTRCEEGNRNKCAEYWPSMEEGT--RAFG--DVVVK--INQH-KRCPDYII PTPRD/hPTPdelta|P23468|1357-1612 EQRSATVVMMTKLEERSRVKCDQYWPS--RGT--ETHG--LVQVT--LLDT-VELATYCV PTPRE/hPTPepsilon|P23469|135-394 EQKSATIVMLTNLKERKEEKCHQYWPD--QGC--WTYG--NIRVC--VEDC-VVLVDYTI PTPRF/hLAR|P10586|1352-1607 EQRTATVVMMTRLEEKSRVKCDQYWPA--RGT--ETCG--LIQVT--LLDT-VELATYTV PTPRG/hPTPgamma|P23470|848-1119 EQNTGIIVMITNLVEKGRRKCDQYWPT--ENS--EEYG--NIIVT--LKST-KIHACYTV PTPRH/hSAP1|Q9HD43|820-1079 EQQSHTLVMLTNCMEAGRVKCEHYWPLDSQPC---THG--HLRVT--LVGE-EVMENWTV PTPRJ/hDEP1|Q12913|1041-1298 EKNVYAIIMLTKCVEQGRTKCEEYWPSKQA-Q---DYG--DITVA--MTSE-IVLPEWTI PTPRK/hPTPkappa|Q15262|887-1141 QEQSACIVMVTNLVEVGRVKCYKYWPD---DT--EVYG--DFKVT--CVEM-EPLAEYVV PTPRM/hPTPmu|P28827|900-1154 HENTASIIMVTNLVEVGRVKCCKYWPD---DT--EIYK--DIKVT--LIET-ELLAEYVI PTPRN/hIA2|Q16849|709-969 ESGCTVIVMLTPLVEDGVKQCDRYWPD--EGA--SLYH--VYEVN--LVSEHIWCEDFLV PTPRN2/hIA2beta|Q92932|745-1005 ESGCVVIVMLTPLAENGVRQCYHYWPD--EGS--NLYH--IYEVN--LVSEHIWCEDFLV PTPRO/hGLEPP1|Q16827|938-1195 QQKSQIIVMLTQCNEKRRVKCDHYWPFTEEPI---AYG--DITVE--MISE-EEQDDWAC PTPRQ/hPTPS31|Q9UMZ3|2036-2292 ETRAKTLVMLTQCFEKGRIRCHQYWPEDNKPV--TVFG--DIVIT--KLME-DVQIDWTI PTPRR/hPCPTP1|Q15256|393-647 QEDSPVIVMITKLKEK-NEKCVLYWPE---KR--GIYG--KVEVL--VISV-NECDNYTI PTPRS/hPTPsigma|Q13332|1393-1648 EQRSATIVMMTRLEEKSRIKCDQYWPN--RGT--ETYG--FIQVT--LLDT-IELATFCV PTPRT/hPTPrho|O14522|889-1143 QENSASIVMVTNLVEVGRVKCVRYWPD---DT--EVYG--DIKVT--LIET-EPLAEYVI PTPRU/hPTPlambda|Q92729|888-1144 QEHCSSIVMITKLVEVGRVKCSRYWPE---DS--DTYG--DIKIM--LVKT-ETLAEYVV PTPRZ/hPTPzeta|P23471|1717-1992 EHNVEVIVMITNLVEKGRRKCDQYWPA--DGS--EEYG--NFLVT--QKSV-QVLAYYTV PTPN1/hPTP1B|P18031|3-277 EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKE--MIFEDTNLKLT--LISE-DIKSYYTV PTPN2/hTCPTP|P17706|5-275 QQKTKAVVMLNRIVEKESVKCAQYWPTD-DQE--MLFKETGFSVK--LLSE-DVKSYYTV PTPN3/hPTPH1|P26045|646-901 DQKLSLIVMLTTLTERGRTKCHQYWPDPPDVM---NHG--GFHIQ--CQSE-DCTIAYVS PTPN4/hMEG1|P29074|655-911 EQGSSMVVMLTTQVERGRVKCHQYWPEPTGSS---SYG--CYQVT--CHSE-EGNTAYIF PTPN5/hSTEP|P54829|300-555 QEHTPIIVMITNIEEM-NEKCTEYWPE---EQ--VAYD--GVEIT--VQKV-IHTEDYRL PTPN6/hSHP1|P29350|244-515 QENSRVIVMTTREVEKGRNKCVPYWPEVGMQR---AYG--PYSVT--NCGE-HDTTEYKL PTPN7/hHePTP|P35236|97-350 QEEVSLIVMLTQLREG-KEKCVHYWPT---EE--ETYG--PFQIR--IQDM-KECPEYTV PTPN9/hMEG2|P43378|303-574 EQKVLVIVMTTRFEEGGRRKCGQYWPLEKDSR--IRFG--FLTVT--NLGV-ENMNHYKK PTPN11/hSHP2|Q06124|247-521 QENSRVIVMTTKEVERGKSKCVKYWPDEYALK---EYG--VMRVR--NVKE-SAAHDYTL PTPN12/hPEST|Q05209|28-293 EYNVVIIVMACREFEMGRKKCERYWPLYGEDP--ITFA--PFKIS--CEDE-QARTDYFI PTPN13/hPTPBAS|Q12923|2213-2467 EQKSTVIAMMTQEVEGEKIKCQRYWPNILGKT---TMV--SNRLRLALVRM-QQLKGFVV PTPN14/hPTPD2|Q15678|909-1180 EQGVNVIAMVTAEEEGGRTKSHRYWPKLGSKHSSATYG--KFKVTTKF--R-TDSVCYAT PTPN18/hBDP1|Q99952|26-291 EFGVKVILMACREIENGRKRCERYWAQEQE-P--LQTG--LFCIT--LIKEKWLNEDIML PTPN21/hPTPD1|Q16825|896-1167 EQGIAIIAMVTAEEEGGREKSFRYWPRLGSRHNTVTYG--RFKITTRF--R-TDSGCYAT PTPN22/hLyPTP|Q9Y2R2|24-289 EYSVLIIVMACMEYEMGKKKCERYWAEPGEMQ--LEFG--PFSVS--CEAE-KRKSDYII PTPN23/hHDPTP|Q9H3S7|770-1130 EQKVSVIVMLVSEAEMEKQKVARYFPTERGQP--MVHG--ALSLA--LSSV-RSTETHVE

S5

PTPRA/hPTPalpha|P18433|241-501 RKFCIQQV-------GDMTNRKPQRLITQFHFTSWPDFGVPFTP--IGMLKFLKKVKACN PTPRB/hPTPbeta|P23467|1703-1963 REFKICGEE----------QLDAHRLIRHFHYTVWPDHGVPETT--QSLIQFVRTVRDYI PTPRC/hCD45|P08575|651-910 QKLNIVNKK----------EKATGREVTHIQFTSWPDHGVPEDP--HLLLKLRRRVNAFS PTPRD/hPTPdelta|P23468|1357-1612 RTFALYKN-------G----SSEKREVRQFQFTAWPDHGVPEHP--TPFLAFLRRVKTCN PTPRE/hPTPepsilon|P23469|135-394 RKFCIQPQ-------LPD-GCKAPRLVSQLHFTSWPDFGVPFTP--IGMLKFLKKVKTLN PTPRF/hLAR|P10586|1352-1607 RTFALHKS-------G----SSEKRELRQFQFMAWPDHGVPEYP--TPILAFLRRVKACN PTPRG/hPTPgamma|P23470|848-1119 RRFSIRNTKVKKGQKGNPKGRQNERVVIQYHYTQWPDMGVPEYA--LPVLTFVRRSSAAR PTPRH/hSAP1|Q9HD43|820-1079 RELLLLQVE----------EQK-TLSVRQFHYQAWPDHGVPSSP--DTLLAFWRMLRQWL PTPRJ/hDEP1|Q12913|1041-1298 RDFTVKNIQ----------TSES-HPLRQFHFTSWPDHGVPDTT--DLLINFRYLVRDYM PTPRK/hPTPkappa|Q15262|887-1141 RTFTLERR-------G----YNEIREVKQFHFTGWPDHGVPYHA--TGLLSFIRRVKLSN PTPRM/hPTPmu|P28827|900-1154 RTFAVEKR-------G----VHEIREIRQFHFTGWPDHGVPYHA--TGLLGFVRQVKSKS PTPRN/hIA2|Q16849|709-969 RSFYLKNVQ-----------TQETRTLTQFHFLSWPAEGTPAST--RPLLDFRRKVNKCY PTPRN2/hIA2beta|Q92932|745-1005 RSFYLKNLQ-----------TNETRTVTQFHFLSWYDRGVPSSS--RSLLDFRRKVNKCY PTPRO/hGLEPP1|Q16827|938-1195 RHFRINYAD----------E---MQDVMHFNYTAWPDHGVPTANAAESILQFVHMVRQQA PTPRQ/hPTPS31|Q9UMZ3|2036-2292 RDLKIERHG----------D---CMTVRQCNFTAWPEHGVPENS--APLIHFVKLVRASR PTPRR/hPCPTP1|Q15256|393-647 RNLVLKQGS-------------HTQHVKHYWYTSWPDHKTPDSA--QPLLQLMLDVEEDR PTPRS/hPTPsigma|Q13332|1393-1648 RTFSLHKN-------G----SSEKREVRQFQFTAWPDHGVPEYP--TPFLAFLRRVKTCN PTPRT/hPTPrho|O14522|889-1143 RTFTVQKK-------G----YHEIRELRLFHFTSWPDHGVPCYA--TGLLGFVRQVKFLN PTPRU/hPTPlambda|Q92729|888-1144 RTFALERR-------G----YSARHEVRQFHFTAWPEHGVPYHA--TGLLAFIRRVKAST PTPRZ/hPTPzeta|P23471|1717-1992 RNFTLRNTKIKKG---SQKGRPSGRVVTQYHYTQWPDMGVPEYS--LPVLTFVRKAAYAK PTPN1/hPTP1B|P18031|3-277 RQLELENLT-----------TQETREILHFHYTTWPDFGVPESP--ASFLNFLFKVRESG PTPN2/hTCPTP|P17706|5-275 HLLQLENIN-----------SGETRTISHFHYTTWPDFGVPESP--ASFLNFLFKVRESG PTPN3/hPTPH1|P26045|646-901 REMLVT-----NT------QTGEEHTVTHLQYVAWPDHGVPDDS--SDFLEFVNYVRSLR PTPN4/hMEG1|P29074|655-911 RKMTLF-----NQ------EKNESRPLTQIQYIAWPDHGVPDDS--SDFLDFVCHVRNKR PTPN5/hSTEP|P54829|300-555 RLISLKSGT-------------EERGLKHYWFTSWPDQKTPDRA--PPLLHLVREVEEAA PTPN6/hSHP1|P29350|244-515 RTLQVSPL----D------NGDLIREIWHYQYLSWPDHGVPSEP--GGVLSFLDQINQRQ PTPN7/hHePTP|P35236|97-350 RQLTIQYQE-------------ERRSVKHILFSAWPDHQTPESA--GPLLRLVAEVEESP PTPN9/hMEG2|P43378|303-574 TTLEIHNT-----------EERQKRQVTHFQFLSWPDYGVPSSA--ASLIDFLRVVRNQQ PTPN11/hSHP2|Q06124|247-521 RELKLSKVGQALL------QGNTERTVWQYHFRTWPDHGVPSDP--GGVLDFLEEVHHKQ PTPN12/hPEST|Q05209|28-293 RTLLLEFQN-------------ESRRLYQFHYVNWPDHDVPSSF--DSILDMISLMRKYQ PTPN13/hPTPBAS|Q12923|2213-2467 RAMTL-----EDI------QTREVRHISHLNFTAWPDHDTPSQP--DDLLTFISYMRH-- PTPN14/hPTPD2|Q15678|909-1180 TGLKV-----KHL------LSGQERTVWHLQYTDWPDHGCPEDV--QGFLSYLEEIQSVR PTPN18/hBDP1|Q99952|26-291 RTLKVTFQK-------------ESRSVYQLQYMSWPDRGVPSSP--DHMLAMVEEARRLQ PTPN21/hPTPD1|Q16825|896-1167 TGLKM-----KHL------LTGQERTVWHLQYTDWPEHGCPEDL--KGFLSYLEEIQSVR PTPN22/hLyPTP|Q9Y2R2|24-289 RTLKVKFNS-------------ETRTIYQFHYKNWPDHDVPSSI--DPILELIWDVRCYQ PTPN23/hHDPTP|Q9H3S7|770-1130 RVLSLQFRD-----------QSLKRSLVHLHFPTWPELGLPDSP--SNLLRFIQEVHAHY

S6

PTPRA/hPTPalpha|P18433|241-501 P-------------QYAGAIVVHCSAGVGRTGTFVVIDAMLDMMHTER---KVDVYGFVS PTPRB/hPTPbeta|P23467|1703-1963 N-----------RSPGAGPTVVHCSAGVGRTGTFIALDRILQQLDSKD---SVDIYGAVH PTPRC/hCD45|P08575|651-910 N-------------FFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAEN---KVDVYGYVV PTPRD/hPTPdelta|P23468|1357-1612 P-------------PDAGPMVVHCSAGVGRTGCFIVIDAMLERIKHEK---TVDIYGHVT PTPRE/hPTPepsilon|P23469|135-394 P-------------VHAGPIVVHCSAGVGRTGTFIVIDAMMAMMHAEQ---KVDVFEFVS PTPRF/hLAR|P10586|1352-1607 P-------------LDAGPMVVHCSAGVGRTGCFIVIDAMLERMKHEK---TVDIYGHVT PTPRG/hPTPgamma|P23470|848-1119 M-------------PETGPVLVHCSAGVGRTGTYIVIDSMLQQIKDKS---TVNVLGFLK PTPRH/hSAP1|Q9HD43|820-1079 D-----------QTMEGGPPIVHCSAGVGRTGTLIALDVLLRQLQSEG---LLGPFSFVR PTPRJ/hDEP1|Q12913|1041-1298 K-----------QSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENEN---TVDVYGIVY PTPRK/hPTPkappa|Q15262|887-1141 P-------------PSAGPIVVHCSAGAGRTGCYIVIDIMLDMAEREG---VVDIYNCVK PTPRM/hPTPmu|P28827|900-1154 P-------------PSAGPLVVHCSAGAGRTGCFIVIDIMLDMAEREG---VVDIYNCVR PTPRN/hIA2|Q16849|709-969 R-------------GRSCPIIVHCSDGAGRTGTYILIDMVLNRMAKGV--KEIDIAATLE PTPRN2/hIA2beta|Q92932|745-1005 R-------------GRSCPIIVHCSDGAGRSGTYVLIDMVLNKMAKGA--KEIDIAATLE PTPRO/hGLEPP1|Q16827|938-1195 -------------TKSKGPMIIHCSAGVGRTGTFIALDRLLQHIRDHE---FVDILGLVS PTPRQ/hPTPS31|Q9UMZ3|2036-2292 -------------AHDTTPMIVHCSAGVGRTGVFIALDHLTQHINDHD---FVDIYGLVA PTPRR/hPCPTP1|Q15256|393-647 L-----------ASQGRGPVVVHCSAGIGRTGCFIATSIGCQQLKEEG---VVDALSIVC PTPRS/hPTPsigma|Q13332|1393-1648 P-------------PDAGPIVVHCSAGVGRTGCFIVIDAMLERIKPEK---TVDVYGHVT PTPRT/hPTPrho|O14522|889-1143 P-------------PEAGPIVVHCSAGAGRTGCFIAIDTMLDMAENEG---VVDIFNCVR PTPRU/hPTPlambda|Q92729|888-1144 P-------------PDAGPIVIHCSAGTGRTGCYIVLDVMLDMAECEG---VVDIYNCVK PTPRZ/hPTPzeta|P23471|1717-1992 R-------------HAVGPVVVHCSAGVGRTGTYIVLDSMLQQIQHEG---TVNIFGFLK PTPN1/hPTP1B|P18031|3-277 S-----------LSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLL PTPN2/hTCPTP|P17706|5-275 S-----------LNPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMEKGD---DINIKQVLL PTPN3/hPTPH1|P26045|646-901 -------------V-DSEPVLVHCSAGIGRTGVLVTMETAMCLTERN---LPIYPLDIVR PTPN4/hMEG1|P29074|655-911 -------------AGKEEPVVVHCSAGIGRTGVLITMETAMCLIECN---QPVYPLDIVR PTPN5/hSTEP|P54829|300-555 Q----------QEGPHCAPIIVHCSAGIGRTGCFIATSICCQQLRQEG---VVDILKTTC PTPN6/hSHP1|P29350|244-515 E-----------SLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQ PTPN7/hHePTP|P35236|97-350 E-----------TAAHPGPIVVHCSAGIGRTGCFIATRIGCQQLKARG---EVDILGIVC PTPN9/hMEG2|P43378|303-574 SLAVSNMGARSKGQCPEPPIVVHCSAGIGRTGTFCSLDICLAQLEELG---TLNVFQTVS PTPN11/hSHP2|Q06124|247-521 E-----------SIMDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQ PTPN12/hPEST|Q05209|28-293 E-------------HEDVPICIHCSAGCGRTGAICAIDYTWNLLKAGKIPEEFNVFNLIQ PTPN13/hPTPBAS|Q12923|2213-2467 -------------IHRSGPIITHCSAGIGRSGTLICIDVVLGLISQ---DLDFDISDLVR PTPN14/hPTPD2|Q15678|909-1180 RHTNSML---EGTKNRHPPIVVHCSAGVGRTGVLILSELMIYCLEHN---EKVEVPMMLR PTPN18/hBDP1|Q99952|26-291 G-------------SGPEPLCVHCSAGCGRTGVLCTVDYVRQLLLTQMIPPDFSLFDVVL PTPN21/hPTPD1|Q16825|896-1167 RHTNSTS---D-PQSPNPPLLVHCSAGVGRTGVVILSEIMIACLEHN---EVLDIPRVLD PTPN22/hLyPTP|Q9Y2R2|24-289 E-------------DDSVPICIHCSAGCGRTGVICAIDYTWMLLKDGIIPENFSVFSLIR PTPN23/hHDPTP|Q9H3S7|770-1130 L----------HQRPLHTPIIVHCSSGVGRTGAFALLYAAVQEVEAGN--GIPELPQLVR

S7

PTPRA/hPTPalpha|P18433|241-501 RIRAQRCQMVQTDMQYVFIYQALLEH PTPRB/hPTPbeta|P23467|1703-1963 DLRLHRVHMVQTECQYVYLHQCVRDV PTPRC/hCD45|P08575|651-910 KLRRQRCLMVQVEAQYILIHQALVEY PTPRD/hPTPdelta|P23468|1357-1612 LMRAQRNYMVQTEDQYIFIHDALLEA PTPRE/hPTPepsilon|P23469|135-394 RIRNQRPQMVQTDMQYTFIYQALLEY PTPRF/hLAR|P10586|1352-1607 CMRSQRNYMVQTEDQYVFIHEALLEA PTPRG/hPTPgamma|P23470|848-1119 HIRTQRNYLVQTEEQYIFIHDALLEA PTPRH/hSAP1|Q9HD43|820-1079 KMRESRPLMVQTEAQYVFLHQCILRF PTPRJ/hDEP1|Q12913|1041-1298 DLRMHRPLMVQTEDQYVFLNQCVLDI PTPRK/hPTPkappa|Q15262|887-1141 ALRSRRINMVQTEEQYIFIHDAILEA PTPRM/hPTPmu|P28827|900-1154 ELRSRRVNMVQTEEQYVFIHDAILEA PTPRN/hIA2|Q16849|709-969 HVRDQRPGLVRSKDQFEFALTAVAEE PTPRN2/hIA2beta|Q92932|745-1005 HLRDQRPGMVQTKEQFEFALTAVAEE PTPRO/hGLEPP1|Q16827|938-1195 EMRSYRMSMVQTEEQYIFIHQCVQLM PTPRQ/hPTPS31|Q9UMZ3|2036-2292 ELRSERMCMVQNLAQYIFLHQCILDL PTPRR/hPCPTP1|Q15256|393-647 QLRMDRGGMVQTSEQYEFVHHALCLY PTPRS/hPTPsigma|Q13332|1393-1648 LMRSQRNYMVQTEDQYSFIHEALLEA PTPRT/hPTPrho|O14522|889-1143 ELRAQRVNLVQTEEQYVFVHDAILEA PTPRU/hPTPlambda|Q92729|888-1144 TLCSRRVNMIQTEEQYIFIHDAILEA PTPRZ/hPTPzeta|P23471|1717-1992 HIRSQRNYLVQTEEQYVFIHDTLVEA PTPN1/hPTP1B|P18031|3-277 EMRKFRMGLIQTADQLRFSYLAVIEG PTPN2/hTCPTP|P17706|5-275 NMRKYRMGLIQTPDQLRFSYMAIIEG PTPN3/hPTPH1|P26045|646-901 KMRDQRAMMVQTSSQYKFVCEAILRV PTPN4/hMEG1|P29074|655-911 TMRDQRAMMIQTPSQYRFVCEAILKV PTPN5/hSTEP|P54829|300-555 QLRQDRGGMIQTCEQYQFVHHVMSLY PTPN6/hSHP1|P29350|244-515 MVRAQRSGMVQTEAQYKFIYVAIAQF PTPN7/hHePTP|P35236|97-350 QLRLDRGGMIQTAEQYQFLHHTLALY PTPN9/hMEG2|P43378|303-574 RMRTQRAFSIQTPEQYYFCYKAILEF PTPN11/hSHP2|Q06124|247-521 MVRSQRSGMVQTEAQYRFIYMA---- PTPN12/hPEST|Q05209|28-293 EMRTQRHSAVQTKEQYELVHRAIAQL PTPN13/hPTPBAS|Q12923|2213-2467 CMRLQRHGMVQTEDQYIFCYQVILYV PTPN14/hPTPD2|Q15678|909-1180 LLREQRMFMIQTIAQYKFVYQVLIQF PTPN18/hBDP1|Q99952|26-291 KMRKQRPAAVQTEEQYRFLYHTVAQM PTPN21/hPTPD1|Q16825|896-1167 MLRQQRMMLVQTLCQYTFVYRVLIQF PTPN22/hLyPTP|Q9Y2R2|24-289 EMRTQRPSLVQTQEQYELVYNAVLEL PTPN23/hHDPTP|Q9H3S7|770-1130 RMRQQRKHMLQEKLHLRFCYEAVVRH

S8

Table S1. Parent constructs used for loop insertions and split-proteins.  Protein   Uniprot.  Entry  #   Residues  PTB1B   P18031   1-­‐321  SHP1   P29350   1-­‐597  PTPH1   P26045   628-­‐909  Lyn-­‐N268-­‐L-­‐S269  (Hahn-­‐Site)  

P07948  211-­‐268,  269-­‐512  

Lyn  N-­‐R311-­‐L-­‐3E12  (311-­‐Site)   211-­‐311,  312-­‐512  Lyn-­‐E393-­‐L-­‐D394  (DFG-­‐Site)   211-­‐393,  394-­‐512  PYLcs   Q8VZS8   33-­‐209  

ABIcs*     P49597   126-­‐423  (D143A)  GID1   Q9MAA7   1-­‐345  GAI  

Q9LQT8  1-­‐533  

GAI(92)   1-­‐93          Constructs used in this study verified by sequencing PTP1B-E75-Loop-E76-H6 MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMECGRGGSGGGGSGGGGSVDGGSGGQLEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNHHHHHH* PTP1B-T165-Loop-Q166-H6 MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTCGRGGSGGGGSGGGGSVDGGSGGQLQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNHHHHHH* PTP1B-E186-Loop-S187-H6 MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPECGRGGSGGGGSGGGGSVDGGSGGQLSPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNHHHHHH*

S9

PTP1B-S205-Loop-P206-H6 MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSCGRGGSGGGGSGGGGSVDGGSGGQLPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNHHHHHH* N-PTP1B-E75-Loop-FRB MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMECGRGGSGGGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ* FKBP-Loop-C-PTP1B-E76-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNHHHHHH* N-PTP1B-T165-Loop-FRB MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTCGRGGSGGGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ* FKBP-Loop-C-PTP1B-Q166-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNHHHHHH* N-PTP1B-S205-Loop-FRB MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSCGRGGSGGGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ*

S10

FKBP-Loop-C-PTP1B-P206-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNHHHHHH* SHP1-G313-Loop-P314-H6 MLSRGWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGCGRGGSGGGGSGGGGSVDGGSGGQLPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRKLHHHHHH* SHP1-N402-Loop-G403-H6 MLSRGWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNCGRGGSGGGGSGGGGSVDGGSGGQLGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRKLHHHHHH* SHP1-E425-Loop-P426-H6 MLSRGWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSECGRGGSGGGGSGGGGSVDGGSGGQLPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRKLHHHHHH* SHP1-L443-Loop-P444-H6 MLSRGWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLCGR

S11

GGSGGGGSGGGGSVDGGSGGQLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRKLHHHHHH* N-SHP1-G313-Loop-FRB MLSRGWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGCGRGGSGGGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ* FKBP-Loop-C-SHP1-P314-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRKLHHHHHH* PTPH1-A708-Loop-N709-H6 MDTLEGSMAQLKKGLESGTVLIQFEQLYRKKPGLAITFAKLPQNLDKNRYKDVLPYDTTRVLLQGNEDYINASYVNMEIPAACGRGGSGGGGSGGGGSVDGGSGGQLDINLVNKYIATQGPLPHTCAQFWQVVWDQKLSLIVMLTTLTERGRTKCHQYWPDPPDVMNHGGFHIQCQSEDCTIAYVSREMLVTNTQTGEEHTVTHLQYVAWPDHGVPDDSSDFLEFVNYVRSLRVDSEPVLVHCSAGIGRTGVLVTMETAMCLTERNLPIYPLDIVRKMRDQRAMMVQTSSQYKFVCEAILRVYEEGLVQMHHHHHH* PTPH1-T795-Loop-G796-H6 MDTLEGSMAQLKKGLESGTVLIQFEQLYRKKPGLAITFAKLPQNLDKNRYKDVLPYDTTRVLLQGNEDYINASYVNMEIPAANLVNKYIATQGPLPHTCAQFWQVVWDQKLSLIVMLTTLTERGRTKCHQYWPDPPDVMNHGGFHIQCQSEDCTIAYVSREMLVTNTQTCGRGGSGGGGSGGGGSVDGGSGGQLDIGEEHTVTHLQYVAWPDHGVPDDSSDFLEFVNYVRSLRVDSEPVLVHCSAGIGRTGVLVTMETAMCLTERNLPIYPLDIVRKMRDQRAMMVQTSSQYKFVCEAILRVYEEGLVQMHHHHHH* PTPH1-D817-Loop-S818-H6 MDTLEGSMAQLKKGLESGTVLIQFEQLYRKKPGLAITFAKLPQNLDKNRYKDVLPYDTTRVLLQGNEDYINASYVNMEIPAANLVNKYIATQGPLPHTCAQFWQVVWDQKLSLIVMLTTLTERGRTKCHQYWPDPPDVMNHGGFHIQCQSEDCTIAYVSREMLVTNTQTGEEHTVTHLQYVAWPDHGVPDDCGRGGSGGGGSGGGGSVDGGSGGQLDISSDFLEFVNYVRSLRVDSEPVLVHCSAGIGRTGVLVTMETAMCLTERNLPIYPLDIVRKMRDQRAMMVQTSSQYKFVCEAILRVYEEGLVQMHHHHHH* PTPH1-S835-Loop-E836-H6 MDTLEGSMAQLKKGLESGTVLIQFEQLYRKKPGLAITFAKLPQNLDKNRYKDVLPYDTTRVLLQGNEDYINASYVNMEIPAANLVNKYIATQGPLPHTCAQFWQVVWDQKLSLIVMLTTLTERGRTKCHQYWPDPPDVMNHGGFHIQCQSEDCTIAYVSREMLVTNTQTGEEHTVTHLQYVAWPDHGVP

S12

DDSSDFLEFVNYVRSLRVDSCGRGGSGGGGSGGGGSVDGGSGGQLDIEPVLVHCSAGIGRTGVLVTMETAMCLTERNLPIYPLDIVRKMRDQRAMMVQTSSQYKFVCEAILRVYEEGLVQMHHHHHH* N-PTPH1-A708-Loop-FRB MDTLEGSMAQLKKGLESGTVLIQFEQLYRKKPGLAITFAKLPQNLDKNRYKDVLPYDTTRVLLQGNEDYINASYVNMEIPAACGRGGSGGGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ* FKBP-Loop-C-PTPH1-N709-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLDINLVNKYIATQGPLPHTCAQFWQVVWDQKLSLIVMLTTLTERGRTKCHQYWPDPPDVMNHGGFHIQCQSEDCTIAYVSREMLVTNTQTGEEHTVTHLQYVAWPDHGVPDDSSDFLEFVNYVRSLRVDSEPVLVHCSAGIGRTGVLVTMETAMCLTERNLPIYPLDIVRKMRDQRAMMVQTSSQYKFVCEAILRVYEEGLVQMHHHHHH* NLyn(268)-Loop-FRB MYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNCGRGGSGGGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ* FKBP-Loop-CLyn(269)-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTREEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIEDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQPHHHHHH* NLyn(311)-Loop-FRB MYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTRCGRGGSGGGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ* FKBP-Loop-CLyn(312)-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLEEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIEDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQPHHHHHH* NLyn(393)-Loop-FRB MYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTREEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIECGRGGSG

S13

GGGSGGGGSVDGGSGGQLEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ* FKBP-Loop-CLyn(394)-H6 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLECGRGGSGGGGSGGGGSVDGGSGGQLDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQPHHHHHH* NLyn(393)-Loop-PYLcs MYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTREEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIECGRGGSGGGGSGGGGSVDGGSGGQLDITQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN* ABIcs

*-Loop-CLyn(394)-H6 MVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKCGRGGSGGGGSGGGGSVDGGSGGQLDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQPHHHHHH* NLyn(393)-Loop-ABIcs

* MYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTREEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIECGRGGSGGGGSGGGGSVDGGSGGQLDIVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK* PYLcs-Loop-CLyn(394)-H6 MGTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMNCGRGGSGGGGSGGGGSVDGGSGGQLDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQPHHHHHH*

S14

NLyn(393)-Loop-GID1 MYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTREEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIECGRGGSGGGGSGGGGSVDGGSGGQLDIMAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAEC* GAI(92)-Loop-CLyn(394)-H6 MGMKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPCGRGGSGGGGSGGGGSVDGGSGGQLDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQPHHHHHH* NLyn(393)-Loop-GAI(92) MYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTREEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIECGRGGSGGGGSGGGGSVDGGSGGQLDIMKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNP* GID1-Loop-CLyn(394)-H6 MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAECCGRGGSGGGGSGGGGSVDGGSGGQLDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQPHHHHHH* PYLcs-Loop-NFluc MGTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMNGTGGGSGGGGSGGGGKLEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG*

S15

CFluc-Loop-ABIcs

* MGMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLGLQGGSGGGGSGGGGSVDVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK* ABIcs

*-Loop-NFluc MVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKGTGGGSGGGGSGGGGKLEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG* CFluc-Loop-PYLcs MGMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLGLQGGSGGGGSGGGGSVDTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN* GID1-Loop-NFluc MATMAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAECDILQGTGGGSGGGGSGGGGKLEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG*

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CFluc-Loop-GAI MGMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLGLQGGSGGGGSGGGGSVDMKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPPSSNAEYDLKAIPGDAILNQFAIDSASSSNQGGGGDTYTTNKRLKCSNGVVETTTATAESTRHVVLVDSQENGVRLVHALLACAEAVQKENLTVAEALVKQIGFLAVSQIGAMRKVATYFAEALARRIYRLSPSQSPIDHSLSDTLQMHFYETCPYLKFAHFTANQAILEAFQGKKRVHVIDFSMSQGLQWPALMQALALRPGGPPVFRLTGIGPPAPDNFDYLHEVGCKLAHLAEAIHVEFEYRGFVANTLADLDASMLELRPSEIESVAVNSVFELHKLLGRPGAIDKVLGVVNQIKPEIFTVVEQESNHNSPIFLDRFTESLHYYSTLFDSLEGVPSGQDKVMSEVYLGKQICNVVACDGPDRVERHETLSQWRNRFGSAGFAAAHIGSNAFKQASMLLALFNGGEGYRVEESDGCLMLGWHTRPLIATSAWKLSTN* GAI-Loop-NFluc MKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPPSSNAEYDLKAIPGDAILNQFAIDSASSSNQGGGGDTYTTNKRLKCSNGVVETTTATAESTRHVVLVDSQENGVRLVHALLACAEAVQKENLTVAEALVKQIGFLAVSQIGAMRKVATYFAEALARRIYRLSPSQSPIDHSLSDTLQMHFYETCPYLKFAHFTANQAILEAFQGKKRVHVIDFSMSQGLQWPALMQALALRPGGPPVFRLTGIGPPAPDNFDYLHEVGCKLAHLAEAIHVEFEYRGFVANTLADLDASMLELRPSEIESVAVNSVFELHKLLGRPGAIDKVLGVVNQIKPEIFTVVEQESNHNSPIFLDRFTESLHYYSTLFDSLEGVPSGQDKVMSEVYLGKQICNVVACDGPDRVERHETLSQWRNRFGSAGFAAAHIGSNAFKQASMLLALFNGGEGYRVEESDGCLMLGWHTRPLIATSAWKLSTNDILQGTGGGSGGGGSGGGGKLEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG* CFluc-Loop-GID1 MGMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLGLQGGSGGGGSGGGGSVDMAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAEC*

S17

GAI(92)-Loop-NFluc MGMKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPDILQGTGGGSGGGGSGGGGKLEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG* CFluc-Loop-GAI(92) MGMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLGLQGGSGGGGSGGGGSVDMKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNP*

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Table S2 List of constructs used in this study with corresponding vectors     Vector Protein

      Vector Protein

1   pRSFD PTP1B-E75-Loop-E76-H6  

37   pETDuet PYLcs-Loop-NFluc

2   pRSFD PTP1B-T165-Loop-Q166-H6  

38   pRSFD CFluc-Loop-ABIcs*

3   pRSFD PTP1B-E186-Loop-S187-H6  

39   pETDuet ABIcs*-Loop-NFluc

4   pRSFD PTP1B-S205-Loop-P206-H6  

40   pRSFD CFluc-Loop-PYLcs

5   pRSFD N-PTP1B-E75-Loop-FRB  

41   pETDuet GID1-Loop-NFluc

6   pRSFD FKBP-Loop-C-PTP1B-E76-H6  

42   pRSFD CFluc-Loop-GAI

7   pRSFD N-PTP1B-T165-Loop-FRB  

43   pETDuet GAI-Loop-NFluc

8   pRSFD FKBP-Loop-C-PTP1B-Q166-H6  

44   pRSFD CFluc-Loop-GID1

9   pRSFD N-PTP1B-S205-Loop-FRB  

45   pETDuet GAI(92)-Loop-NFluc

10   pRSFD FKBP-Loop-C-PTP1B-P206-H6  

46   pRSFD CFluc-Loop-GAI(92)

11   pRSFD SHP1-G313-Loop-P314-H6  

47   pcDNA (3.1) + PTP1Bcat-H6

12   pRSFD SHP1-N402-Loop-G403-H6  

48   pcDNA (3.1) + N-PTP1B-S205-Loop-FRB

13   pRSFD SHP1-E425-Loop-P426-H6  

49   pcDNA (3.1) + FKBP-Loop-C-PTP1B-P206-H6

14   pRSFD SHP1-L443-Loop-P444-H6  

50   pcDNA (3.1) + SHP1-FL-H6

15   pRSFD N-SHP1-G313-Loop-FRB  

51   pcDNA (3.1) + N-SHP1-G313-Loop-FRB

16   pRSFD FKBP-Loop-C-SHP1-P314-H6  

52   pcDNA (3.1) + FKBP-Loop-C-SHP1-P314-H6

17   pRSFD PTPH1-A708-Loop-N709-H6  

53   pcDNA (3.1) + Lyn-cat-H6

18   pRSFD PTPH1-T795-Loop-G796-H6  

54   pcDNA (3.1) + NLyn(268)-Loop-FRB

19   pRSFD PTPH1-D817-Loop-S818-H6  

55   pcDNA (3.1) + FKBP-Loop-CLyn(269)-H6

20   pRSFD PTPH1-S835-Loop-E836-H6  

56   pcDNA (3.1) + NLyn(393)-Loop-FRB

21   pRSFD N-PTPH1-A708-Loop-FRB  

57   pcDNA (3.1) + FKBP-Loop-CLyn(394)-H6

22   pRSFD FKBP-Loop-C-PTPH1-N709-H6  

58   pcDNA (3.1) + NLyn(393)-Loop-ABIcs*

23   pRSFD NLyn(268)-Loop-FRB  

59   pcDNA (3.1) + PYLcs-Loop-CLyn(394)-H6

24   pRSFD FKBP-Loop-CLyn(269)-H6        25   pRSFD NLyn(311)-Loop-FRB        26   pRSFD FKBP-Loop-CLyn(312)-H6        27   pRSFD NLyn(393)-Loop-FRB        28   pRSFD FKBP-Loop-CLyn(394)-H6        

29   pRSFD NLyn(393)-Loop-PYLcs        

30   pRSFD ABIcs*-Loop-CLyn(394)-H6

       31   pRSFD NLyn(393)-Loop-ABIcs

*        

32   pRSFD PYLcs-Loop-CLyn(394)-H6        33   pRSFD NLyn(393)-Loop-GID1        34   pRSFD GAI(92)-Loop-CLyn(394)-H6        35   pRSFD NLyn(393)-Loop-GAI(92)        36   pRSFD GID1-Loop-CLyn(394)-H6        

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Figure S2. Effect of rapamycin addition for A) split-PTP1B, B) split-SHP-1, C) split-PTPH1, and D) One inactive fragment (FKBP-N709 of PTPH1) containing a hexa-Histidine tag. Rapamycin has no effect on the background signal for any of the systems tested in this manuscript, whether phosphatases or kinases. However, we often note a small and reproducible drop in the background signal when any inactive hexa-His tagged protein is expressed in the lysate or when the inactive complexes are present in the absence of CID. This small loss in the background signal could potentially be due to displacement of native enzymes present in the lysate by any hexa-His tagged protein as shown in case (D).

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Figure S3. Small molecule dependent luciferase reassembly with the PYLcs/ABIcs*//ABA and

GID1/GAI//GA3 based CID systems. (A) Ligand-gated firefly luciferase (Fluc) reassembly of fusions of Nfluc/CFluc and the PYLcs/ABIcs

*//ABA and GID1/GAI//GA3 CIDs utilizing in vitro transcribed mRNA in a rabbit reticulocyte lysate translation system, in the absence or presence of 1.2 mM ABA or 1.2 mM GA3, respectively.3 (B) A significant signal increase is observed only upon addition of the ligands (ABA and GA3). The best working construct pairs found for each CID system in the context of split-luciferase were ABIcs

*-NFluc/CFluc-PYLcs and GID1-NFluc/CFluc-GAI(92), respectively. All data are presented without normalization as relative luminescent units subtracted from reactions containing only lysate.

S21

Figure S4. The activity of the FKBP/FRB//Rapamycin dependent split-Lyn fragments at three different dissection sites. (A) Topological and three-dimensional representation of the catalytic domain of Lyn kinase (PDB ID 3A4O) showing the sites chosen for fragmenting the kinase. (B) Three-dimensional representation of the different split-Lyn fragments. (C) Kinase activity assay results for the different FKBP/FRB//Rapamycin dependent split-Lyn constructs using the Lyn peptide substrate and 32P-ATP.4 The three split-Lyns show activation only in the presence of rapamycin. All data are presented without normalization as total radioactive counts subtracted from reactions containing only lysate.

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Supplementary Methods PYLcs/ABIcs

*//ABA and GID1/GAI//GA3 association dependent reassembly of firefly luciferase. All luminescent assays were carried out in duplicate as described previously and repeated on different days.4 Briefly, 1 pmol of each construct were co-translated in RRL for 90 minutes at 30˚C in a final volume of 25µL, in the absence or presence of 1.2 mM ABA (PYLcs/ABIcs

* ) or 1.2 mM GA3 (GID1/GAI ). 20 µL of complex and 80 µL of luciferase assay reagent were incubated for 1 min and luminescence was measured using a Turner Biosystem 20/20n luminometer with 10 s integration time. FKBP/FRB//Rapamycin Split Lyn kinase activity assay. All kinase activity assays were carried out in duplicate as described previously and also repeated on different days.3 Briefly, 1 pmol of each construct were co-translated in RRL for 90 min at 30˚C in a final volume of 25µL, in the absence or presence of 250 nM rapamycin. The proteins were purified using Ni-NTA resin, and the kinase assay was then carried out in a final volume of 30 µL. Protein on resin was incubated with a peptide substrate (Lyntide: EDPIYEFLPAKKK at 24 µM) and Kinase Reaction Buffer (supplemented with 250 nM rapamycin) for 30 min at RT. 10 µL of radioactive ATP mix (100 µM, 1/24 32P-ATP, 23/24 31P-ATP) was added to the protein substrate mix and incubated for 4 hours at room temperature. The reaction was spotted on P81 paper and dried. The P81 paper was washed for 3 min 3x with 500 mL of 0.85% phosphoric acid and 1x with 500 mL of acetone. The P81 paper was immersed in 10 mL of scintillation cocktail and the radioactive counts measured. References:    (1) Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T. J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Soeding, J.; Thompson, J. D.; Higgins, D. G. Molecular Systems Biology 2011, 7. (2) Goujon, M.; McWilliam, H.; Li, W.; Valentin, F.; Squizzato, S.; Paern, J.; Lopez, R. Nucleic Acids Research 2010, 38, W695. (3) Porter, J. R.; Stains, C. I.; Jester, B. W.; Ghosh, I. Journal of the American Chemical Society 2008, 130(20), 6488–6497. (4) Camacho-Soto, K.; Castillo-Montoya, J.; Tye, B, Ghosh, I. Journal of the American Chemical Society 2014, 136(10), 3995–4002.