ABCC8

Transcription

>> GOOD AFTERNOON, EVERYONE. IT'S MY PLEASURE TO WELCOME YOU TO THE WEDNESDAY AFTERNOON LECTURE. TODAY TO BE DELIVERED BY DOUGLAS REES, WHO IS THE ROSCOE GILKE DICKINSON PROFESSOR AT THE CALIFORNIA INSTITUTE OF TECHNOLOGY, ALSO AN INVESTIGATOR OF THE HOWARD HUGHES MEDICAL INSTITUTE. IN MAY OF 1989, I HAD THE EXPERIENCE AFTER SEARCHING FOR THE GENE RESPONSIBLE FOR CYSTIC FIBROSIS, WORKING WITH MY COLLEAGUES IN TORONTO THAT WE ACTUALLY FOUND THE ANSWER. SO THE CDNA SEQUENCE WAS QUICKLY TRANSLATED INTO A PROTEIN AND THE PROTEIN WAS COMPARED -- YEAH, 1989, YOU COULD STILL DO THIS STUFF, TO OTHER KNOWN PROTEINS, AND TO OUR GREAT BENEFIT, IT TURNED OUT THAT THIS WAS NOT A PROTEIN OF INSCREUTABLE AMINO ACID SEQUENCE, IT ACTUALLY CLEARLY HAD THE CHARACTERISTICS OF A ATP BINDING CASSETTE AND ABC TRANSPORTER. IT TURNED OUT THAT JACK RIORDAN, ONE OF THE COLLEAGUES, HAD BEEN WORKING ON THAT FIELD FOR A COUPLE OF YEARS. IN THAT BRIEF COUPLE OF WEEKS, BASED UPON THAT INFORMATION THAT WAS KNOWN AT THAT POINT, WERE ABLE TO MAKE SOME STATEMENTS ABOUT THIS PROTEIN'S STRUCTURE MIGHT LOOK LIKE AND SOMETHING ABOUT HOW IT MUT MIGHT FUNCTION ALTHOUGH THERE WERE MANY QUESTIONS ABOUT WHAT ITS ROLE MAY BE, TURNED OUT IT WAS A TRANSPORTER OF CHLORIDE. SO I BECAME FAMILIAR A LITTLE BIT, THAT'S QUITE A LONG TIME AGO, WITH THIS WHOLE FIELD THAT YOU'RE ABOUT TO HEAR ABOUT FROM AN ABSOLUTE EXPERT, SO I'M LOOKING FORWARD TO HEARING WHAT DOUGLAS HAS TO TELL US ABOUT THE WORK HE HAS DONE ON ABC TRANSPORTERS. THIS IS ONE OF THOSE WONDERFUL FAMILIES OF PROTEINS THAT CLEARLY EVOLUTION HAS ENJOYED USING FOR ALL KINDS OF APPLICATIONS OVER THE COURSE OF 2 OR 3 BILLION YEARS. HIS PARTICULAR ACADEMIC HISTORY, I WILL JUST QUICKLY MENTION. UNDERGRADUATE AT YALE, PH.D. FROM HARVARD, AND THEN FOLLOWING A POSTDOC AT MINNESOTA, WENT TO THE UCLA, WHERE HE WAS ON THE FACULTY FOR SEVEN YEARS UNTIL MOVING TO CAL TECH, WHERE HE'S BEEN ZIPS 1989. SINCE 1989. IT'S A GREAT PLEASURE TO WELCOME HIM HERE FOR THE WEDNESDAY AFTERNOON LECTURE. PLEASE JOIN ME IN WELCOMING DR. DOUGLAS REES. [APPLAUSE] >> THANK YOU FOR YOUR VERY KIND INTRODUCTION. IT'S A REAL PLEASURE TO BE HERE TODAY AND GET TO EXPERIENCE A LITTLE WINTER BEFORE RETREATING BACK WEST. SO THIS AFTERNOON, JUST A QUICK OUTLINE OF PREVIEW OF COMING ATTRACTIONS. THERE WILL BE THREE PARTS TO THE TALK, A BRIEF INTRODUCTION ON ABC TRANSPORTERS, AND THEN TWO STORIES. ONE IS SORT OF AN ONGOING STORY ON THE E. COLI THIAMINE ABC IMPORTER. THEN I'LL END WITH SOME MORE RECENT WORK ON AN ATM1 BACTERIAL ABC EXPORTER. SO JUST BY WAY OF INTRODUCTION, THE PASSAGE OF VIRTUALLY ALL MOLECULES IN AND OUT OF CELLS IS MEDIATED BY SPECIALIZED INTEGRAL MEMBRANE PROTEINS KNOWN AS CHANNELS OR TRANSPORTERS. SO WE'VE BEEN PARTICULARLY INTERESTED IN ONE CLASS OF TRANSPORTERS, THE ATP BINDING CASSETTE OR ABC SUPERFAMILY OF TRANSPORTERS THAT USE THE BINDING AND HYDROLYSIS OF ATP TO TRANSLOCATE A WIDE RANGE OF MOLECULES IN AND OUT OF CELLS. SO ABC TRANSPORTERS, AT LEAST THE FIRST ORDER, COME IN TWO FLAVORS. THERE ARE IMPORTERS WHICH ARE FOUND IN BACTERIA AND ARCHEA THAT WILL IMPORT NUTRIENTS INTO CELLS, AND THEN THERE ARE EXPORTERS THAT ARE FOUND IN ALL KINGDOMS OF LIFE THAT HAVE A VERY WIDE SUBSTRATE SPECIFICITY, THEY CAN PUMP ANTICANCER DRUGS OUT OF CELLS, AND SO ON. THE FAMILY OF TRANSPORTERS ARE RECOGNIZED BY A CHARACTERISTIC FOUR-DOMAIN CHARACTERIZATION. THEY HAVE TWO COPIES EACH OF THE ATP BINDING COULD SET OR THE CONSERVED NUCLEOTIDE BINDING DOMAINS, AND THIS WAS THE WAY WHEN FRANCIS DISCOVERED THE CFTR GENE, THEY COULD ASSIGN CFTR TO AN ABC TRANSPORTER FAMILY BECAUSE OF THE PRESENCE OF THESE SEQUENCE MOTIFS. THERE ARE THEN TWO COPIES OF TRANSMEMBRANE SPANNING DOMAINS, AND THESE ARE NOT UNIVERSALLY CONSERVED ACROSS THE FAMILY. AS WE'LL SEE THAT THERE ARE CURRENTLY THREE FAMILIES OF ABC TRANSPORTERS THAT ARE RECOGNIZED RECOGNIZED. ALTHOUGH THE FOLDS OF THESE THREE FAMILIES ARE DISTINCT, THEY DO HAVE A COMMON STRUCTURAL ELEMENT KNOWN AS A COUPLING HELIX, WHICH SERVES AS THE INTERFACE BETWEEN THE TRANSMEMBRANE DOMAINS AND THE NUCLEOTIDE BINDING DOMAINS. FOR IMPORTERS LIKE THE ME THIGH NEEN IMPORTER, THERE'S A PERIPLASMIC BINDING PROTEIN THAT'S RESPONSIBLE FOR SUBSTRATE SPECIFICITY, DELIVERS IT TO THE TRANSPORTER. AND THEN IN ADDITION, THERE MAY BE REGULATORY DOMAINS THAT COULD BE FUSED TO EITHER THE ABC OR THE TRANSMEMBRANE DOMAINS THAT ARE ABLE TO MODULATE THE ACTIVITY OF THE TRANSPORTER. SO THE ATP BINDING CASSETTES ARE REALLY SERVING AS THE ENGINE THAT DRIVES SUBSTRATE TRANSLOCATION, AND SO THE NUCLEOTIDE BINDING HAS THESE CONSERVED -- SUCH AS THE WALKER A MOTIF OR P-LOOP, FOUND IN MANY DIFFERENT KINDS, THERE'S A SIGNATURE MOTIF, SOMETIMES KNOWN AS LSGGQ FOR THE AMINO ACID SEQUENCE, WHICH IS A CHARACTERISTIC OF THIS PARTICULAR FAMILY, AND THEN THERE ARE ADDITIONAL DOMAINS SUCH AS THE WALKER-B Q OF H LOOP AND SO ON. NOW INDIVIDUAL SUBUNITS ARE UNABLE TO FIND NUCLEOTIDES, BUT NUCLEOTIDE BINDING IS A PROPERTY OF THE DIMER OF NUCLEOTIDE BINDING SUBSTRATES, SO THE NUCLEOTIDES SITTING AT THE INTERFACE BETWEEN TWO NUCLEOTIDE BINDING DOMAINS SORT OF SANDWICHED BETWEEN THE WALKER-A AND THE SIGNATURE MOTIF OR THE TWO SUBUNITS. SO NUCLEOTIDE ATP HYDROLYSIS REQUIRES A CLOSED INTERFACE OF THESE TWO SUBUNITS IN ORDER TO CAT ATP HYDROLYSIS, THEN IT'S THE PRODUCTS ASSOCIATE -- RELEASE -- THEN THERE WILL BE REARRANGEMENT IN THE RELATIVE ORIENTATIONS OF THE TWO SUBUNITS WHICH WILL DRIVE THE CONFORMATIONAL CHANGE IN THE TRANSMEMBRANE DOMAIN. NOW BECAUSE THE NUCLEOTIDE BINDING SITES ARE REALLY REMOTE FROM THE TRANSMEMBRANE DOMAIN BUT IT'S NOT A DIRECT INTERACTION, BUT INSTEAD THE IND INT ACTION IS MEDIATED BY THE COUPLING HELICES, WHICH WOULD COME IN BETWEEN TWO TRANSMEMBRANE HELICES HERE. NOW CURRENTLY, THERE ARE THREE FAMILIES OF ABC TRANSPORTERS RECOGNIZED. FOR THE IMPORTERS, TYPES 1 AND 2, AND THEN THE EXPORTER FAMILY, SO THE TYPE 2 IMPORTERS IN THE FIRST STRUCTURAL REPRESENTATION OF AN ABC CONTRIBUTO TRANSPORTER WAS THE THIAMINE B12 IMPORTER WHICH WAS SOLVED BY A POSTDOC IN MY GROUP. CASPER THEN WENT TO AN INDEPENDENT CAREER IN ZURICH WHERE HE DETERMINED THE FIRST TYPE 1 TRANSPORTER FOR -- AS WELL AS THE FIRST ABC EXPORTER, SAV1866. SO I'LL BE TALKING ABOUT THE ME THIGH NEEN TRANSPORTER AND THEN SOME REASON WORK ON THIS ATM1 MIC WHICH WAS EXPORTER FAMILY. JUST LIKE TO MAKE, I GUESS, THREE ADDITIONAL POINTS. ONE IS PROBABLY AT LEAST FOR IMPORTERS, THE BEST CHARACTERIZED ABC TRANSPORTER SYSTEM IS THE MALTOSE SYSTEM, THIS IS REALLY THROUGH THE GROUP OF -- AND AMY DAVIDSON AT PURDUE WHERE A NUMBER OF INTERMEDIATE STEPS IN THE TRANSPORT CYCLE HAVE BEEN CAPTURED CRYSTALOGRAPHICALLY. SECOND POINT IS ALMOST ALL OF THESE ARE BACTERIAL KRPTERS. TRANSPORTERS, THE EXCEPTION BEING THE MOUSE P GLYCOPROTEIN, DETERMINED BY JEFFREY CHANG, ALSO A POSTDOC IN MY GROUP. EUKARYOTIC, ESPECIALLY MAMMALIAN ABC TRANSPORTERS HAVE STILL BEEN A REALLY DIFFICULT PROBLEM. WE SHOULD PROBABLY ADD, I GUESS AS A BRANCH, MORE DISAT THAT PARTICULAR TIME BRANCH OF THIS FAMILY, THESE ENERGY COUPLING FACTOR TRANSPORTERS, WHICH ARE FOUND IN BACTERIA WHERE STRUCTURES WERE RECENTLY DETERMINED BY A GROUP IN BEJING. THESE HAVE THE SAME TYPE OF KNEW NUCLEOTIDE BINDING DOMAIN DIMER, BUT THEY HAVE A VERY DISTINCTIVE ARRANGEMENT OF THE TRANSMEMBRANE DOMAIN, SO THEY'RE NOT OBVIOUSLY IN THIS GROUP, BUT IN TERMS OF THE ENGINE, IT'S THE SAME SORT OF PROCESS. ABC TRANSPORTERS LIKE MANY TRANSPORTERS ARE THOUGHT OF AS WORKING THROUGH AN ALTERNATING ACCESS MECHANISM, WHERE THERE'S AN OUTWARD FACING CONFORMATION AND AN INWARD FACING CONFORMATION, AND THE TRANSITION BETWEEN THE TWO IS DRIVEN BY BINDING OF ATP HYDROLYSIS AS WELL AS COUPLING TO THE DELIVERY OR BINDING OF THE SUBSTRATE. SO IT'S BELIEVED THAT WHILE THESE ABC TRANSPORTERS HAVE, I'D SAY, SORT OF THE SAME TYPE OF CHEMISTRY THAT'S TAKING PLACE IN TERMS OF ATP BINDING AND HYDROLYSIS AND HAVING TWO CONFORMATIONAL STATES, I THINK THE DETAILS, OF COURSE, CAN VARY SIGNIFICANTLY. NOTHE NOT ONLY BETWEEN IMPORTERS AND EXPORTERS BUT EVEN WITHIN FAMILIES. THEY DON'T WORK ALL IN IDENTICAL FASHION. NOW THE WAY IN WHICH WE'VE BEEN APPROACHING THIS PROBLEM, AT LEAST INITIALLY, WAS THAT WE ARE REALLY INTERESTED IN TRYING TO GET A STRUCTURAL CHARACTERIZATION OF NACB TRANSPORTER, AND THE EXACT ONE WASN'T PROBABLY SO CRITICAL. SO WE TOOK BASICALLY THIS FUNNEL APPROACH WHERE WE STARTED WITH CLONING A NUMBER OF HOMOLOGUES, WE FIGURED FOR EVERY ONE HOMOLOGUE, THERE WAS A REASONABLE CHANCE IT WOULDN'T WORK, BUT IF WE TRIED TWO HOMOLOGUES, THERE WAS A MORE FAVORABLE CHANCE ONE OF THOSE WOULD WORK, AND IF YOU KEEP TRYING ENOUGH HOMOLOGUES, THEN THEY'LL MAKE IT THROUGH THIS SORT OF FUNNEL WHERE YOU GET -- LOSE SOME OF THE EXPRESSION, SOME OF THE CRYSTALLIZATION, BUT THEN IF YOU TRIED ENOUGH, THEN ONE WOULD WORK. SO THIS IS IF THE -- THE PROBABILITY OF THIS HAPPENING IS GREATER THAN ZERO. OF COURSE IF THE PROBABILITY IS EITHER NEGATIVE OR IMAGINARY, THEN THIS IS A LESS HAPPY OUTCOME. AND THIRDLY, IN OUR EXPERIENCE WITH EUKARYOTIC PROTEINS, THIS HAS BEEN THE SORT OF REGIME WE'VE BEEN OPERATING IN. BUT THAT'S JUST US. IT'S NOT A FUNDAMENTAL PROBLEM, INTRINSIC PROBLEM WITH THE SYSTEM. NOW THIS HAS BEEN CHARACTERIZED IN MANY WAYS. THE ONE IS SHOT ON GOALS, THE MORE TIMES YOU TRY TO SCORE, THE MORE LIKELY IT IS THAT YOU'LL SCORE. ANOTHER I'M TOLD IS SPEED DATING, WHERE YOU JUST BASICALLY WANT TO SORT OF LOOK AT A POTENTIAL CANDIDATE LONG ENOUGH TO KNOW WHETHER YOU WANT TO KEEP PURSUING THIS OR MOVE ON TO THE NEXT ONE. NOW I AM AT A TECHNICAL INSTITUTE, THE CALIFORNIA INSTITUTE OF TECHNOLOGY, SO EVERYTHING IS STATE OF THE ART. SO THAT'S TRUE FOR CRYSTALLIZATION. THIS CASE, OUR ROBOT IS NAMED ALLEN LEE. SO MOST OF WHAT WE'VE DONE IN THE MEMBRANE PROTEIN WORLD HAS REALLY COME OUT OF HIS PIPE HEADER. SO MOST OF MY JOB IN GOING PLACES, I CAN TALK ABOUT SCIENCE, I MERELY WANT TO SAY WHAT A GREAT JOB ALLEN IS DOING. SO THE FIRST OF TWO STORIES. I'LL DESCRIBE SOME WORK ON THE ME THIGH NEEN ABC IMPORTER. SO THIS WAS ORIGINALLY IDENTIFIED BY ROBERT KADNER AS A HIGH AFFINITY IMPORTER OF L AND D METHIONINE. IF YOU'RE INTERESTED USING SELENOMETHIONINE, THIS IS THE TRANSPORT SYSTEM THAT TAKES THAT UP FOR LABELING FROM AD PHASING. SO KADNER, THIS IS WITH CELL-PHORESIS TEMS BUT HE DID THIS REALLY BEAUTIFUL SET OF STUDIES SHOWING -- INTRIGUINGLY HE FOUND THAT AS THE LEVEL OF INTRACELLULAR ME THIGH KNEE INCREASED, THE RATE OF TRANSPORT DECREASED, AND THAT THE KINETICS OF THIS PROCESS AND THE PROPERTIES OF THIS PROCESS WERE CONSISTENT WITH THIS REGULATION ACTING AT THE LEVEL OF TRANSPORTER AS OPPOSED TO TRANSCRIPTION OR TRANSLATION. THIS IS KNOWN AS TRANSINHIBITION WHERE INCREASED -- DECREASE THE RATE OF UPTAKE FROM THE OTHER SIDE OF THE MEMBRANE. NINA AND ALLEN, MY GROUP SOLVED THE STRUCTURE OF THE METHIONINE IMPORTER FROM E. COLI, AND FOUND AT THIS POINT THAT IT HAD 5 TRANSMEMBRANE HELICES, WHICH SORT OF UNEXPECTEDLY, AT LEAST AT THE TIME, RESEMBLED A CORSET OF HELICES THAT HAD BEEN FOUND IN TWO PREEMSLY DETERMINED STRUCTURES. ONE WAS THE MA -- THAT CASPER HAD DETERMINED THAT HAD SIX HELICES, THEN THE -- THAT -- SO THIS IS LIKE A STRIPPED DOWN VERSION OF A TYPE 1 IMPORTER WITH THIS FIVE TRANSMEMBRANE HELICES. PER SUBUNIT. SO IT'S A RANGE INWARD FACING CONFORMATION WITH A WIDELY SEPARATED SET OF NUCLEOTIDE BINDING DOMAINS. REMEMBER THE NUCLEOTIDE BINDING DOMAINS HAVE TO COME TOGETHER TO HYDROLYZE ATP, SO PRESUMABLY THIS IS A CONVERSATION THAT CAN'T HYDROLYZE ATP. IN ADDITION, FUSED TO THE C-TERMINUS OF THE NUCLEOTIDE BINDING DOMAINS WITH AN EXTRA DOMAIN WHICH DIMERIZED, WHICH WE BELIEVED WERE A REGULATORY DOMAIN, AND OUR INITIAL EVIDENCE FOR THAT IS THAT WE COULD SHOW EITHER THROUGH BIOSYNTHETIC LABELING OR JUST ADDING SLEE KNOW METHIONINE TO CHRIS CAL TALLS, THAT THERE WAS A BINDING SITE FOR METHIONINE ON THESE DOMAINS. SO THIS WAS SUGGESTIVE THAT -- IT MAY HAVE SOMETHING TO DO WITH THE MECHANISM OF TRANSINHIBITION. INTERACTS WITH THREE DIFFERENT TYPES OFLY AGAINST, S OF LIGANDS. ONE IS RELATED TO THE INTERACTION WITH ATP AND ADP, AND WHETHER OR NOT YOU NEED TWO ACTIVE FUNCTIONAL -- SITES TO HYDROLYZE ATP. METHIONINE IN THE SITE OF TRANSINHIBITION, METHIONINE ALSO COMES IN AS A SUBSTRATE BOUND TO THE BINDING PROTEIN MET Q, AND WE'VE DONE A LOT OF WORK AND ARE ABLE TO FINALLY PREPARE A STABLE COMPLEX OF MET Q AND MET NI BUT WE HAVEN'T BEEN ABLE TO SOLVE THE STRUCTURE OF IT SO THIS THIRD PART WILL BE LEFT FOR A FUTURE SEMINAR. SO METNI IN THE PRESENCE OF DETERGENT HYDROLYZES ATP. THAT MAY NOT SOUND SURPRISING SINCE IT IS AN ATPASE BUT IT ALWAYS STRUCK ME AS AN ODD REACTION BECAUSE THERE'S NO SUBSTRATE IN THIS REACTION, SO THIS WOULD BE AN EXAMPLE OF UNCOUPLED ATPASE ACTIVITY. WHETHER THIS IS BECAUSE THE TRANSPORTER IS DETERGENT SOLUBLIZE AND NOT THE CONFORMATIONAL ENERGETICS THE SAME AS IN THE MEMBRANE OR WHETHER THESE TRANSPORTERS JUST HAVE A BASAL ATPASE RATE, I'M NOT SURE. IF YOU LOOK AT LOW CONCENTRATION, YOU'LL SEE THERE'S EVIDENCE OF SIG MOI DAL COOPERATIVE INTERACTION, AND IF YOU FIT SORT OF JUST A BASIC TWO-SITE GENETIC MODEL TO EXTRACT OUT BINDING CON ASSISTANTS, THERE'S EVIDENCE FOR POSITIVE COOPERATIVITY, WHERE THE SECOND ATP BINDS MORE TIGHTLY THAN THE FIRST. THE RATE OF ATP HYDROLYSIS IS ABOUT 25 PER MINUTE. NOW THE INTACT WILD TYPE TRANSPORTER HAS TWO IDENTICAL ATPASE SITES INTERFACE BETWEEN THE TWO END SUBUNITS, AND SO WE ARE INTERESTED IN WHETHER OR NOT THEY WERE BOTH REQUIRED FOR ATP ACTIVITY. AND SO BECAUSE THE WAY THIS TRANSPORTER IS PRODUCED, WE HAVE TWO IDENTICAL COPIES OF MET I AND TWO OF THE NATPASE, YOU CAN'T JUST MUTATE ONE OF THE TWO SITES, BUT WE'VE SET UP A DOUBLE TAGGED SYSTEM WHERE WE HAD ONE SUBUNIT WITH A -- AND ANOTHER WITH A FLAG TAG, AND WE COULD MUTATE ONE VERSION, HAVE WILD TYPE FOR THE OTHER, AND WE COULD SELECT FOR CHIMERAS THAT CONTAINED BOTH ONE WILD TYPE AND ONE MUTATION. THIS E166Q MUTATION IS IN THE WALKER-B MOTIF. IT IS OFTEN USED IN ABC TRANSPORTERS TO REALLY REDUCE BUT NOT COMPLETELY ELIMINATE ATP HYDROLYSIS. SO YOU MIGHT EXPECT SEVERAL THINGS. YOU MIGHT EXPECT IF YOU MEU TAKE ONMUTATEONE ACTIVE SITE, YOU'LL GET ALL ATPASE ACTIVITY AND NO TRANSPORT. SO THIS IS EXTRAPOLATED OR GENERALIZED FROM A CERTAIN SET OF EXPORTERS WHERE THERE'S TWO NON-EQUIVALENT SITES TO BEGIN WITH. SO THIS IS IN LIKE THE TAP TRANSPORTER CFTR, IF YOU MUTATE THE DEGENERATE SITE, PROBABLY CAN'T HIDIZE ATP, IT WORKS FINE. I LEARNED THIS MORNING THAT IN -- YOU GET NO ATPAASE ACTIVITY WHEN YOU MUTATE ONE SITE. THEN THERE'S THE PRECEDENT FROM THE HISTADINE TRANSPORTER -- HALF ATP AIS AND -- THAT MUST BE P GLYCOPROTEIN BUT IT'S ALSO THE EXPERIENCE OF THE MALL TOES TRANSPORTER, AND THE B12 UPTAKE SYSTEM. WE FIND THERE'S ESSENTIALLY NO CHANGE IN ACTIVITY, THAT THE BINDING CON ASSISTANTS AND THE KINETIC CONSTANTS ARE MORE OR LESS THE SAME. SO THIS KIE AMERICA MUTANT WITH ONLY ONE ACTIVE SITE STILL -- BASICALLY THE SAME RATES AS THE WILD TYPE CONSTRUCT. WHAT WE DON'T KNOW YET, BECAUSE WE'VE HAD PROBLEMS GETTING OUR TRANSPORT ASSAY TO WORK, IS WHETHER OR NOT THESE WILL ACTUALLY TRANSPORT. BUT THIS IS CONSISTENT WITH A MODEL PROPOSED FOR SOME TRANSPORTERS WHERE YOU DON'T HAVE TWO CONCERTED ATP HYDROLYSIS EVENTS, BUT YOU ONLY HYDROLYZE ONE ATP AT A TIME. SO THIS IS SUGGESTIVE THAT THAT'S WHAT'S HAPPENING HERE, AND IT DOESN'T ACTUALLY HAVE TO ALTERNATE BETWEEN SITES. THE OTHER ASPECT I'D LIKE TO DISCUSS BRIEFLY IS THE MECHANISM OF TRANSINHIBITION, SO THE HYPOTHESIS WOULD BE THAT IF YOU BIND A LIGAND TO THIS REGULATORY DOMAIN, THAT IT WILL SOMEHOW BASICALLY TURN OFF THE ENGINE, AND THE WAY IN WHICH YOU MIGHT HYPOTHESIZE IT COULD TURN OFF THE ENGINE IS BY BASICALLY HOLDING -- OR STAIRICALLY SEPARATING THE TWO NUCLEOTIDE BINDING DOMAINS SO THAT THEY'RE UNABLE TO COME TOGETHER AND FORM THE FORM THAT'S PRODUCTIVE PER ATP HYDROLYSIS. NOW UNFORTUNATELY, SORT OF THE EXPERIMENT OF SHOWING THAT THE SEPARATION BETWEEN THE SUBUNIT CAN BE CORRELATED OR ANTICORRELATED WITH BOUND LIGAND, WE HAVEN'T BEEN ABLE TO DO, BECAUSE WE HAVEN'T BEEN ABLE TO EVER GET SORT OF A FORM CRYSTALLIZED WITH THE TWO SUBUNITS TOGETHER. WHAT WE DO HAVE IS SOME EVIDENCE IN DIFFERENT DETERGENTS THAT INDICATE THAT AT LEAST THERE CAN BE CHANGES IN HOW THESE TWO TERMINAL DOMAINS ARE POSITIONED WITH RESPECT TO THE NUCLEOTIDE BINDING DOMAIN, AND THAT LIGAND BINDING THEN COULD SHIFT THIS EQUILIBRIUM EITHER IN THE FAVOR OF THE OUTWARD -- THE SEPARATED CONFORMATION OR THE ONE WHERE THEY'RE TOGETHER. NOW THE TIME WE WERE FIRST CHARACTERIZING MET NI, THEY FOUND A VERY SIMILAR PHENOMENON IN HIS SYSTEM WITH A DISTINCT TYPE OF REGULATORY DOMAIN, BUT HE WAS ABLE TO SHOW THAT TONGUE STATE BINDING TO THE C TERMINAL DOMAIN LED TO A WIDELY SEPARATED CONFORMATION OF THE BIND DOMAINS, AGAIN CONSISTENT WITH THIS TRANSINHI BAITION WORKING BY THE REGULATORY DOMAIN STERICAL LFT Y PREVENTING THE NBDs FROM COMING TOGETHER. MORE RECENTLY IN THE MALTO SYSTEM, THEY WERE ABLE TO SHOW FOR CARBON KA TAB LIGHT REPRESSION THAT THIS A2 PROTEIN IS NOT A LIGAND, BUT IT BASICALLY HAS THE SAME EFFECT OF STABILIZING THE INWARD FACING CONFORMATION WHERE WHERE THE NBDs ARE SEPARATED. SO WE HAVE DONE KINETIC STUDIES SHOWING IF WE ADD LMD METHIONINE, THEY SERVE AS NON-COMPETITIVE INHIBITORS, WHICH IS WHAT WE WOULD EXPECT SINCE METHIONINE WILL INHIBIT ATP HYDROLYSIS, BUT THEY'RE NOT COMPETING FOR BINDING TO THE SAME SITES, AND THERE DOES NOT SEEM TO BE ANY DIFFERENCE BETWEEN THE WILD TYPE AND THE CHIMERIC VARIANTS HERE. IN TERMS OF THE CONFORMATIONAL CHANGES TAKING PLACE, AGAIN, WE DON'T HAVE THIS DIRECTLY COMPLETELY WORKED OUT IN TERMS OF HOW METHIONINE BINDING DOES THIS, BUT IN THE COURSE OF LOOKING AT DIFFERENT VARIANTS, WE'VE SEEN THAT THERE ARE TWO DIFFERENT CONFORMATIONS OF THIS REGULATORY DOMAIN THAT DIFFER BY THE REGISTER OF HYDROGEN BONDING BETWEEN THE BETA SHEET BETWEEN THESE TWO STRANDS. SO ESSENTIALLY THIS IS WORKING AS A SORT OF T TOGGLE SWITCH WHEN LIGAND BINDS, THERE'S A CHANGE IN CONFORMATION LIKE A TWO-STATE SYSTEM. THE WAY IN WHICH THIS MAY OCCUR IS THAT THE ALLOSTERIC LIGAND, IN THIS CASE -- MA THIGH NEEN BINDING TO HAD DOMAIN BINDS AT A SITE WHICH IS ADJACENT, WHICH IS FORMED BY BETA STRANDS ALONG THIS INTERFACE, AND SORT OF AMAZINGLY IN THE ABSENCE OF -- YOU HAVE THE SIDE CHAINS OF TWO OTHER ME THINE KNEES FROM THE PROTEINS, 301 AND 312, THAT ARE PRESENT. IT SEEMS THAT THE LIGAND COMES IN AND IT ACTUALLY THE SUL FAR DISPLACES THE SIDE CHAIN OF METHIONINE 301, AND THAT DISPLACEMENT MUST THEN SERVE TO DRIVE THIS REARRANGEMENT IN THE CONFORMATIONS OF THE REGULATORY DOMAIN. OKAY. SO IF IT WE NOW SHIFT GEARS FROM THE IMPORTERS TO THE EXPORTER STORY, SO WE BECAME REALLY INTERESTED IN AN ABC EXPORTER ATM1 THROUGH OBSERVATIONS OF ROLAND WILL, WHO DEMONSTRATED IN YEAST THAT THE YEAST VERSION ATM1, OR ATM1 IS THE YEAST PROTEIN, IS A MITOCHONDRIAL ABC EXPORTER INDICATED IN BIOSYNTHESIS. SINCE THAT'S PART OF MY GROUP THAT'S REALLY INTERESTED IN IRON SULFUR PROTEINS, THIS SEEMED LIKE SORT OF A PERFECT SYSTEM TO BE WORKING ON. OKAY. IT WAS SHOWN THAT IF YOU HAVE A DEFICIENCY IN ATM1 IN YEAST, THEY ACCUMULATE IRON IN THE MY TOMITOCHONDRIA, THEY CAN'T MAKE CYTOPLASMIC SULFUR CONTAINING PROTEINS. THE HUMAN HOMOLOGUE IS ABCB7, MUTATIONS IN THIS HAVE BEEN FOUND TO CAUSE ANEMIA. AND THE SUBSTRATE IS NOT KNOWN. BUT FOR REASONS WE'LL SEE, IT MAY INCLUDE GLUE TOE THIGH OWN OR POSSIBLY SOME PRECURSORS IN BIOSYNTHESIS. THIS IS A NUMBER OF RELATED PROTEIN FAMILIES HERE SO IT'S A MEMBER OF A SUPERFAMILY WHICH INCLUDES ABC B6, WHICH IS INVOLVED IN PORPHYRIN IMPORT, THEN THESE HEAVY METAL TOLERANCE FACTORS ARE ALL PART OF THIS FAMILY, AND THE SEQUENCE IDENTITY IS AROUND 40%. SO THESE ARE, I'D SAY, PRETTY CLOSE RELATIVES HERE. AND JUST TO KEEP IN MIND, THIS IS THE YEAST ATM1, HERE'S THE HUMAN ABCB7, AND OVER HERE IS THE STRUCTURE I'LL BE DESCRIBING. WELL, SORT OF THE PARADIGM OF AN ABC EXPORTER IN TERMS OF ARCHITECTURE IS THIS BACTERIAL PROTEIN ASB1866, A HALF TRANSPORTER WHERE A SUBUNIT HAS A FEW TRANSMEMBRANE DOMAIN IN THE NUCLEOTIDE BINDING DOMAIN, SO THE YELLOW AND THE GREEN SUBUNIT. IN COMPARISON TO IMPORTERS, THE EXPORTERS HAVE THESE REALLY LONG INTRACELLULAR LOOPS OR HELICAL EXTENSIONS. THE LOOPS BETWEEN TWO OF THESE EXTENDED HELICES, ICL1, THAT GOES BETWEEN TM2 AND 3, THEN ICL2 WHICH GOES BETWEEN 4 AND 5, INTERACT WITH DIFFERENT -- EACH INTERACTS WITH A DIFFERENT NUCLEOTIDE BINDING DOMAIN. BUT THESE ARE SERVING AS THE COUPLING HELICES THAT HAVE BEEN SEEN IN THE IMPORTERS. SO THIS IS THE TYPE OF ABC TRANSPORTER THAT WE HAVE IN SEVEN FAMILIES, ABCB1 P-GLYCOPROTEIN,ABCC1 MRP1. ONE THING THAT HAD BEEN MISSING THROUGH THESE STRUCTURALLY CHARACTERIZED ABC TRANSPORTERS IS A HIGH RESOLUTION CHARACTERIZATION OF HOW SUBSTRATES ARE BINDING TO THE TRANSPORTER. SO IT WAS KNOWN ROUGHLY THROUGH WORK ON P-GLYCOPROTEIN WHERE THE BINDING SITES ARE, BUT THEY HADN'T BEEN DEFINED SORT OF HIGH RESOLUTION. OKAY. SO JONAS LEE WAS A POSTDOC IN MY GROUP, AND TOOK THIS FUNNEL APPROACH TO CRYSTAL SCREENING. ABOUT 10 YEARS AGO, WE TRIED VARIOUS EUKARYOTIC HOMOLOGUES, AND THAT FUNNEL ENDED, I'D SAY, PRETTY ABRUPTLY AND UNSATISFYINGLY. SO ANYWAY, HE STARTED WITH 40, 34 CLONES, 18 EX-PRESSORS, SIX BEHAVED WELL, DETERGENTS ARE IMPORTANT. SO DETERGENTS LOOK PROMISING, SO 42 CONSTRUCTS, THREE CRYSTALS, AND NO GOOD DEFRACK TORES. SO THIS IS WHERE THE FUNNEL SYSTEM STARTS HAVING A LITTLE PROBLEM. ONE OF THE ONES THAT LOOKED MOST PROMISING IN TERMS OF BEING FAIRLY GIVING, NICE-LOOKING CRYSTALS, WHICH IN REAL STAYS SPACE ISN'T REALLY VERY RELEVANT, BUT IT MAKES YOU FEEL LIKE YOU'RE MAKING PROGRESS, WAS THIS ONE, NAATM1. SO JONAS TRIED, I'D SAY FOR A YEAR, ALL OF HIS CLEVER ENGINEERING THINGS, BUYING SURFACE RESIDUES, TRUNCATIONS, ALL SORTS OF THINGS. AND JUST KEPT GETTING BEAUTIFUL CRYSTALS THAT DEFRACTURED ABOUT 10 ANGSTROMS RESOLUTION. I THINK YOU CAN ONLY UNDERSTAND THAT SORT OF ARISES FROM THIS SORT OF DESPERATION THAT'S REALLY BORNE OF DESPAIR AND FRUSTRATION, AND IT HE TOOK SOME ADVICE FROM -- OR HE TOOK SERIOUSLY THIS SORT OF INNOCENT QUESTION ASKED BY A ROTATION STUDENT, AND THE BACKGROUND OF THIS WAS, OF COURSE, WE DO THESE SCREENS TRYING TO FIND THE BEST DETERGENT, AND THE BEST DETERGENT MEANS THAT YOU CAN SOL BULLIZE THE PROTEIN FROM THE MEMBRANE AND THE PROTEIN REMAINS MONO DISPERSED WHEN IT'S GOING THROUGH A -- COLUMN OR LIGHT SCATTERING OR WHATEVER. AND HERE'S SOME EXAMPLES. SO DDM, THIS IS AN SDS GEL, SO SOMETHING LIKE BDM, THIS IS A GOOD DETERGENT. SOMETHING LIKE PEGA11, THIS IS A BAD DETERGENT. SO YOU COULD CLASSIFY DETERGENTS IN DIFFERENT GROUPS. PROBABLY GOOD, BAD AND UGLY, GOOD, OKAY, BAD, AND USUALLY, THEN, YOU TAKE THE GOOD ONES AND YOU OPTIMIZE THE GOOD ONES. AND SO THAT GOT THE SORT OF 10 ANGTROMS RESOLUTION. WHY DON'T YOU TRY MIXTURES OF BAD DETERGENTS SM SO O, SO OF COURSE ANYONE WOULD KNOW YOU WOULDN'T TRY BAD DETERGENTS BECAUSE THEY'RE BAD DETERGENTS, BUT NOTHING ELSE WAS WORKING SO JONAS SOMEHOW DECIDE TO TAKE A MIXTURE OF FOUR BAD DETERGENTS AND WHAT HE GOT WORKED REALLY WELL. SO THIS IS NOW THIS GROUP NUMBER TWO HERE, WHERE YOU SEE HE GETS REALLY GREAT EXTRACTION, AND IN TERMS OF THE PROPERTIES, NOT ONLY DID THEY EXTRACT WELL, BUT HE WAS GETTING A NON-AGGREGATED PEAK ON THE SIZE EXCLUSION COLUMN, AND SO BASICALLY IT SEEMED THAT TWO OF THESE DETERGENTS WERE NEEDED TO HAVE SORT OF A MONO DISPERSE PEAK. THE OTHER TWO WERE REQUIRED FOR CRYSTALLIZATION. ANYWAY, THIS WAS THE MIXTURE HE USED FOR EXTRACTION AND CRYSTALLIZATION, AND ULTIMATELY WAS ABLE TO SOLVE THE STRUCTURE AT 2.4 ANGSTTROMS RESOLUTION. WELL REFINED STRUCTURE. INWARD FACING CONFORMATION. SO NOT SURPRISINGLY IT LOOKS MORE LIKE SAV1866 IN TERMS OF THE BASIC ARCHITECTURE, TWO HALF TRANSPORTERS OR TWO SUBUNITS EACH WITH SIX TRANSMEMBRANE HELICES, THE SEPARATED NBDs, C TERMINAL EXTENSION WHICH FORMS SOME INTERFACE. AND THIS IS JUST THE CARTOON VERSION. WE NOW HAVE THE STRUCTURE OF A TRANSPORTER BUT KNOW NOTHING ABOUT ITS FUNCTION IN VIVO OR IN VITRO. SO ONCE AGAIN, WE FOLLOW THE OBSERVATIONS OF ROLAND WILL ON THE YEAST ATM1 WHERE HE WAS ABLE TO SHOW THAT GLUE TOE THIGH HOME DISTRIBUTIVE -- WE ALSO USED LIGAND STIMULATED ATPASE ACTIVITY AS A PROXY FOR TRANSPORT ACTIVITY. AND WERE ABLE TO FIND THOSE DERIVATIVES WORKED AS WELL. TRY PEPTIDE IN TWO OXIDATION STATES REDUCED AND OXIDIZED. IT'S GOT THIS GAMMA GLUTAMATE, SO WITH THESE AMINO -- PREAMINO -- AND CAR BOX ILL GROUP, THE GLIE SEEN. SO THIS ALSO FUNCTIONS IN DETOXIFICATION TO REACTIVITY OF THE -- WITH VARIOUS -- AND METALS AND SO ON. SO BASICALLY ATP ACE ACTIVITY IS STIMULATED BY THINGS LIKE WHEN WE ADD MERCKURATED GLUTE THIGH OWN, WE GET MUCH BETTER BINDING OF THIS SYSTEM. IT WORKS WITH BOTH OXIDIZED AND REDUCED GLUTATHIONE. THEN JANET YAN GWAS ABLE TO SHOW WE COULD DEMONSTRATE UPDATE TAKE OF OXIDIZED GLUE TATHIONE -- WE HAD TROUBLE RECONSTITUTING THE PURE PROTEIN, SO THIS IS JUST WHATEVER MEMBRANE PROTEINS ARE IN E. COLI OVER OH EXPRESSING -- BUT WE WERE ABLE TO SHOW IN THIS CASE THE PROTEIN CAN TRANSPORT OXIDIZED GLUE TATHIONE. IN TERMS OF IN VIVO ACTIVITY, IT'S STILL NOT CLEAR TO ME WHAT IT'S DOING IN THE NATIVE ORGAN, BUT -- WAS ABLE TO SHOW WHEN HE INTRODUCED THIS INTO VARIOUS METAL SENSITIVE E. COLI STRAINS AND OVEREXPRESSED THE PROTEIN, NOT REALLY OVEREXPRESSED, THIS WAS ABLE TO CONFER PROTECTION AGAINST BOTH SILVER AND MERCURY. SO WE THINK THIS IS PROBABLY PLAYING SOME SORT OF ROLE IN DETOXIFICATION, AT LEAST MERCURY AND SILVER TOXICITY, AND PERHAPS OTHER TYPES OF GLUE TA THIONE CONJUGATES. THERE'S JUST SORT OF A RELATIVELY RECENT DISCUSSION OF USING SILVER, CONTINUING AN OLD DISCUSSION OF SILVER OF AN ANTIMICROBIAL ABOUT HOW IT'S WORKING, AND I GUESS ONE THING THAT'S CLEAR, NO MATTER HOW IT'S WORKING, BACTERIA WILL FIND SOME WAY TO GET AROUND THIS PROBLEM N THIS CASE PROBABLY BY EXPRESSING E FLUX PUMPS. JONAS WAS ABLE TO ESTABLISH WHERE THE BINDING SITES FOR GLUTATHIONE WERE IN THE STRUCTURE, AND WE'LL SEE THERE WERE TWO SITES SITTING SORT OF -- THIS IS INWARD FACING CONFORMATION, SO TOWARDS THE CYTOPLASMIC SIDE. IN ADDITION, THERE WERE TWO BOUND MOLECULES OF LDAO. ONE WAS A DETERGENT. ONE OF THE THINGS WHICH I WONDERED ABOUT, WE HAVE NO DIRECT EVIDENCE, IS WHETHER THE WAY THESE TWO GLUTATHIONES ARE POSITIONED, THESE ARE TWO OXIDIZED GLUTATHIONES OR FOUR HALF GLUE TA THIG THIGH OWE OWNS, THEY WOULD BE COMPATIBLE WITH THE -- IRON SULFUR CLUSTER. ACTUALLY JIMMY COWAN IN A PAPER THAT'S JUST COME OUT ONLINE HAS SOME RESULTS OR INTERPRETIVES CONSISTENT WITH THAT MODEL. THE LDAO MODEL, ON THE TWO SUBUNITS, WHICH IS THAT COUPLING -- ONE OF THE COUPLING HELICES BETWEEN THE TRANSMEMBRANE DOMAIN AND THE NUCLEOTIDE BINDING DOMAIN, AND PRESUMABLY IN SOME WAYS MUST BE SORT OF INTERFERING WITH THE CONFORMATIONAL CHANGES REQUIRED FOR ATPASE ACTIVITY, SO IF YOU INCREASE THE LDAO CONCENTRATION, THE RATE OF ATP HYDROLYSIS DECREASES. OKAY. JUST A LOOK AT THE BINDING SITES IN MORE DETAIL, THIS IS FOR -- THE PRIMARY SITE AND THE SECONDARY SITE. THE GLUTATHIONE OXIDIZES, IT SPANS THE SUBUNIT INTERFACES, BUT THE PRIMARY INTERACTIONS INVOLVE HELICES 5 AND 6 OF 1 SUBUNIT, THEN THE OTHER SUBUNIT. THERE'S ALSO SOME INTERACTIONS WITH TM3 AND WITH T4. THE SECONDARY SITE IS A MORE ARGININE-RICH ENVIRONMENT. ONE MUTATION IN THE HUMAN PROTEIN CORRESPONDS WITH THE -- CORRESPONDS TO WHAT WOULD BE 324, SO WHETHER THAT SOMEHOW PERTURBS THE SUBSTRATE BINDING SITE IS NOT CLEAR, BUT IT'S SUGGESTIVE FROM THIS IT STRUCTURE. THEN WE'VE STARTED DOING MUTAGENESIS EXPERIMENTS, SHOWING WHEN WE ME MUTATE, SO IN THIS CASE, THERE'S INTERACTIONS BETWEEN SORT OF THE AMINO AND CORE BOX ILL GROUPS OF THE GAMMA GLUE FOMPLING THE STRONG HYDROGEN BONDING INTERACTIONS. WHEN WE MUTATE THESE RESIDUES, WE CAN HAVE QUITE A RANGE OF EFFECTS ON THE ATPASE ACTIVITY, BUT IN CONTRAST TO THE WILD TYPE, WHERE THERE'S A SUBSTANTIAL -- THREE OR FOUR FOLD INCREASE IN THE ATPASE ACTIVITY IN ADDING GLUTATHIONE, SO WHEN WE MUTATE THE RESIDUES INTERACTING WITH THE GLUTATHIONE, WE ELIMINATE -- -- WE ELIMINATE THE GLUTATHIONE STIMULATION BUT THEN WE CAN EITHER ELIMINATE THE ATPASE ACTIVITY OR THIS 195 THAT INDIRECTLY IS INTERACTING WITH THE RESIDUE, SORT OF THE SECOND SITE. WE SEE WE CAN GET HYPERACTIVITY AS WELL. SO WE STILL DO UNDERSTAND IN COMPLETE DETAIL THE CONSEQUENCES OF THESE MUTATIONS, BUT THEY DO AFFECT THE ACTIVITY. NOW, WHAT WE WOULD LIKE TO BE ABLE TO DO IS TO RELATE THE ATPASE ACTIVITY IN THE SUBSTRATE BINDING, AND SO IN TERMS OF THE CONFORMATIONAL CHANGES THAT ARE REQUIRED TO GO FROM THE OUTWARD TO INWARD. IF ONE LOOKS AT WHAT HAPPENS IN THE RANGE OF STRUCTURES GOING FROM THE INWARD FACING TO THE OUTWARD FACING CONFORMATION, IT'S CLEAR THAT THERE'S NOT A -- GOING IN THIS RNA ACCESS MECHANISM, THERE'S NOT A RIGID BODY MOVEMENT OF THE SUBUNITS, BUT INSTEAD THERE'S -- FOR SIGNIFICANT REARRANGEMENTS WITHIN A SUBUNIT, AND THESE HAVE TWO TYPES, ONE IS GOING TO THE INWARD FACING CONFIRMATION, TM4 AND 5, SPLIT AWAY FROM THE OTHER FOUR TRANSMEMBRANE HELICES IN THE SAME SUBUNIT, 1, 2, 3 AND 6, WHEREAS THE IN THE OUTWARD FACING CONFORMATION, IT'S NOW ONE AND TWO SPLIT AWAY FROM 3, 4, 5 AND 6. SO WHILE THERE'S NOT A RIGID BODY CHANGE IN THE SUBUNIT CONFORMATION, YOU CAN ACTUALLY IDENTIFY THE STRUCTURALLY CONSERVED ELEMENTS WITHIN THE TRANSMEMBRANE DOMAIN, WHICH WOULD BE TM1 AND 2, 3 AND 6, AND 4 AND 5. THERE'S A FOURTH STRUCTURALLY CONSERVED ELEMENT AS WELL, WHICH IS THE NUCLEOTIDE BINDING -- WHICH I HAVEN'T SHOWN HERE. THIS HIGHLIGHTS A RANGE IN -- THIS TRANSPORTER, LIKE MANY OTHER MEMBRANE PROTEINS, HAS EVIDENCE FOR AN INTERNAL SYMMETRY, WHERE THERE'S A TWOFOLD ACCESS. YOU COULD IMAGINE THAT GOES BETWEEN 3 AND 6 SO ESSENTIALLY HELICES 1, 2, 3, WITH 4, 5 AND 6. SO SORT OF A COMPARABLE THINGS HAVE BEEN SEEN IN MAJOR FACILITATOR SUPERFAMILY TRANSPORTERS, LUCY HAS WORKED OUT THIS BEAUTIFUL MECHANISM OF A TRANSPORTER FUNCTION BY USING THALITY MATING ACCESS WITH ITS INTERNAL SYMMETRY TO GUIDE MODELING OF OUTWARD AND INWARD FACING CONFORMATION. IF WE DO A DIFFERENT TYPE OF REPRESENTATION WHERE INSTEAD OF JUST SUPER IMPOSING ON CELLS, WE TAKE THE DIFFERENT STRUCTURALLY CHARACTERIZED ABC EXPORTERS AND WE SUPERIMPOSE THEM ALL ON 3 AND 6, WE SEE THAT THERE CAN BE QUITE A RANGE OF RELATIVE ORIENTATIONS BETWEEN ESPECIALLY 4 AND 5, AS WELL AS 1 AND 2 WITH RESPECT TO THIS COURSE. THIS IS SAYING THERE'S NOT A RINL ID BODY CHANGE, BUT WE DO HAVE A SUBUNIT THAT'S MADE UP OF THE STRUCTURALLY CONSERVED ELEMENTS BUT THE JOINTS BETWEEN THEM ARE MORE FLEXIBLE, AND THIS WOULD BE AN EXAMPLE OF AN ARTICULATED DESIGN. SIGNIFICANTLY THE LIGAND BINDING SITES SPAN THESE CONSERVATIVE ELEMENTS, SO THE GLUTATHIONE IS INTERACTING ESPECIALLY WITH 6 -- 3, 6, 4 AND 5. WE DON'T ACTUALLY SEE MUCH INTERACTIONS WITH 1 AND 2, BUT THERE ARE VERY STRONG INTERACTIONS OF THE LIGANDS GOING BETWEEN HELICES 6 AND 5 SO YOU COULD IMAGINE IN THE PRESENCE OR ABSENCE OF THE LIGANDS, YOU CAN CHANGE THE RELATIVE ORIENTATION OF THESE INTERACTIONS. OKAY. SO WHAT HAVE WE LEARNED FROM THESE STUDIES? WELL, ABC TRANSPORTERS, ALTHOUGH THEY DO HAVE A CHARACTERISTIC NUCLEOTIDE BINDING MOTIF, THAT HAS BEEN USED TO IDENTIFY MEMBERS OF THIS FAMILY, THEY ARE STRUCTURALLY DIVERSE WITH AT LEAST THREE DISTINCT TRANSMEMBRANE FOLDS AND I THINK THE DETAILED MECHANISM WILL SPECIALIZE IN PARTICULAR TRANSPORTERS. ONE WAY YOU CAN THINK ABOUT A TRANSPORTER WORKING IS IT'S BASICALLY LIKE AN AIR LOCK, WHERE AN AIR LOCK HAS TWO GATES AND IF YOU OPEN UP THOSE GATES AT THE SAME TIME, THEN YOU HAVE A PROBLEM, EVERYTHING WOULD GO DOWN THE -- FAVORABLE DIRECTION. BUT IF UL CAN CONTROL THE SEQUENCE OF GATES OPENING AND CLOSING, THEN YOU CAN MOVE THINGS FROM A REGION OF LOW CONCENTRATION TO HIGH CONCENTRATION HENCE THE ROLE OF ATP BINDING AND HYDROLYSIS TO REALLY CONTROL THAT SEQUENCE OF GATE OWN OAPING AN OPENING AND CLOSING. ONE WAY WHICH REGULATORY DOMAINS MAY WORK TO INHIBIT TRANSPORT OR, I GUESS, ACTIVATE IT, IS TO REGULATE THE ASSOCIATION OF THE ABC SUBUNIT. AT LEAST IN IMPORTERS, THIS SEEMS TO BE A PREFERRED OR DOMINANT WAY OF REGULATING ACTIVITY TO PREVENT UP TAKE IN THE PRESENCE OF SUFFICIENT NUTRIENTS. THIS EXPORTER LIKELY PLAYS A ROLE IN CELLULAR DETOXIFICATION PROCESSES BY SUPPORTING GLUE TA THIGH ON DERIVATIVES, THEN -- WHERE THERE'S CONSERVED ELEMENTS WITH FLEXIBLE JOINTS AND LIGANDS SPAN BETWEEN THOSE ELEMENTS TO CONTROL THE RELATIVE POSITIONS IN THE CONFORMATION OF THE DIFFERENT STATES ASSOCIATED WITH ALTERNATING ACCESS. TO ACKNOWLEDGE THE SPECIFIC PEOPLE THAT WERE DOING THIS WORK, THE WORK NATM1 WAS DONE BY TWO POSTDOCS, FORMER POSTDOC DID THE IN VIVO ASSAY. THE WORK ON -- WAS ORIGINALLY DONE BY ERIC JOHNSON WITH ALAN, THEN -- SUBSEQUENTLY. FINALLY, SINCE I HAVE THIS OPPORTUNITY, I SHOULD REALLY TAKE THIS TO ACKNOWLEDGE, I'VE HAD THE GREAT FORTUNE OF BEING ABLE TO WORK ON MEMBRANE PROTEINS FOR NEARLY 30 YEARS, STARTING WITH WORK ON THE REACTION CENTER WITH GEORGE AND JIM ALLEN, TODD YATES, THROUGH TO THE PRESENT, AND SO I'VE BEEN REALLY FORTUNATE, I HAVE A GREAT GROUP OF STUDENTS, POSTDOCS, COLLABORATORS. THIS WORK HAS BEEN ONLY POSSIBLE THEN THROUGH THE RESOURCES OF FACILITIES SUCH AS -- SOURCES, ALS, SUPPORT OF THE MOORE FOUNDATION, THE -- AND CERTAINLY BY FUNDING BY NIH, HHMI, AND I'M VERY APPRECIATIVE OF HAVING HAD THIS OPPORTUNITY, AND WOULD LIKE TO THANK YOU AND ALL THESE PEOPLE FOR THEIR EFFORTS. THANKS. [APPLAUSE] >> WE HAVE TIME FOR QUESTIONS. THERE ARE MICROPHONES IN THE AISLES. PLEASE USE THOSE SO THAT PEOPLE WATCHING ON THE VIDEO CAN HEAR THE QUESTIONS. >> SO IF YOU EXTRACT WITH DDM BUT THEN EXCHANGE INTO THE MIXTURE OF DETERGENT, DO YOU NOT REPRODUCE THE CRYSTALS OR THE DEFRACTION? IN OTHER WORDS, IS IT NECESSARY TO HAVE THE MIXTURE OF DETERGENT ALL THE WAY FROM THE BEGINNING? >> THAT'S THE WAY WE DID THE EXPERIMENT, SO I CAN'T ANSWER YOUR SPECIFIC QUESTION. WE REALLY TRY HARD TO USE THE SAME DETERGENTS FOR EXTRACTION AND FOR PURIFICATION AND CRYSTALLIZATION. AS DEFINITELY AS POSSIBLE TO EXCHANGE THEM, THAT'S BEEN USED SUCCESSFULLY, MANY OCCASIONS, BUT TO ME, THAT INTRODUCES MORE SORT OF A PHILOSOPHICAL THING THAT INTRODUCES THE POSSIBILITY OF SOME HETEROGENEITY OR EAR REPRODUCIBILITY FROM BATCH TO BATCH, SO I GUESS TO ME, PROBABLY THE ONLY THING THAT'S WORSE THAN NOT GETTING CRYSTALS IS TO GET CRYSTAL ONE TIME. AND NEVER BEING ABLE TO REPRODUCE THEM. SO WE'VE TRIED HARD TO SORT OF MAINTAIN THE STANDARD PROTOCOL, AND DETERGENT -- SWITCHING DETERGENTS HAS BEEN ONE OF THESE THINGS THAT HAS ALWAYS BOTHERED ME. BUT I MEAN, IT SHOULD BE OKAY. >> SURE. >> IT'S EMOTIONAL. >> SO FOLLOW UP TO THAT IS THAT DO YOU SEE ANYTHING BETWEEN YOUR DDM EXTRACTED MATERIAL AND YOUR USING THE FOUR DETERGENT COMBINATION THAT TELLS YOU THERE'S ANY DIFFERENCE IN THE QUALITY OF THE SAMPLES? >> NOT THAT WE'VE SEEN. IT COULD JUST BE SOMETHING TO DO -- I MEAN, SO WE DON'T -- WE DON'T KNOW WHAT THE KEY THING, IT COULD HAVE TO DO WITH THE SIZE OR THE SHAPES OF THE MICE -- OR SOMETHING AND HOW THEY'RE ABLE TO PACK TOGETHER, OR IT COULD HAVE TO DO WITH PROTEIN STABILITY, OR IT COULD HAVE TO DO WITH EXTRACTING SOME COMPONENT FROM THE MEMBRANE LIKE LID IPS OR WHATEVER, SO WE REALLY JUST DON'T KNOW WHAT WAS THE KEY TO THAT SUBSTITUTION. >> IN THE EXPERIMENT IN WHICH YOU SHOWED OXIDIZED GLUTATHIONE UPTAKE IN THE E. COLI MEMBRANE VESICLES, DID YOU LOOK TO SEE IF THE PROTEIN WAS IN THE RIGHT ORIENTATION? >> IT ALMOST CERTAINLY WOULD BE RANDOMIZED. THE WAY WE MADE THE VESICLES WAS TO PASS THEM THROUGH AN EXTRUDER MANY TIMES, SO WE'RE ASSUMING THAT IT'S RANDOMIZED. >> I IF YOU LOOK AT THE EXTRACTORS OF THE EXPORTERS AND IMPORTERS, THE ONLY EVIDENT DIFFERENCE LOOKS LIKE THE LONG INTRACELLULAR LOOPS FOR THE EXPORTER. DO YOU THINK THIS CAN EXPLAIN SOMEHOW THE -- OF HOW THESE TRANSPORTER WORKS OR MAYBE WE ARE MISSING SOMETHING ELSE? >> YEAH, SO RIGHT NOW I'D SAY THAT'S A STRONG CORRELATION, BUT I GUESS TO BE CAREFUL, I'M GUILTY OF THIS, ESPECIALLY, WE CALL THOSE PROTEINS EXPORTERS, BUT THERE HAVE BEEN VERY FEW THAT HAVE ACTUALLY BEEN, I'D SAY, CHARACTERIZED, SO EVEN EXPORTERS, SOME OF THEM MAY BE IMPORTERS. OR THEY 345EU WORK -- I THINK IT REALLY DEPENDS ON SORT OF THE RELATIVE AFFINITIES OF THE SUBSTRATE FOR THE INWARD AND OUTWARD FACING CONFORMATION. THE SAME THINGS WE CALL IMPORTERS, IN SOME CASES, MAYBE THEY'RE WORKING AS EXPORTERS. THEIRS IS PARAPLASMIC BINDING PROTEIN COMPONENT. SO I GUESS FOR THAT SAID, I WAS LISTENING TO ONE OF MY COLLEAGUES TALK ABOUT FOR DEVELOPMENT OF THE HEART AND THE CIRCULATING SYSTEM AROUND THIS WAS, I GUESS, IN SOME MOUSE SYSTEM. IT ACTUALLY LOOKED LIKE PEARSALL TICK PRIME MINISTE PUMPS, AND DEPENDING ON WHERE YOU WERE DOING THE FORCING, YOU COULD GET THE SYSTEM TO RUN IN EITHER DIRECTION. SO IT MAY BE -- I WAS WONDERING IF THERE WERE SOME PARALLELS TO THESE EXPORTERS THAT BASICALLY YOU'RE SORT OF CHANGING THE POSITION OF THE ENGINE THAT'S DRIVING THIS WITH RESPECT TO WHERE THE -- POINT S MAYBE THERE IS SOMETHING TO THAT CORRELATION THAT REALLY IS INTRINSIC, BUT I DON'T HAVE ANY SPECIFIC MODEL TO SAY THAT -- >> THAT MEANS THE QUESTION IS STILL OPEN? >> I THINK SO. YEAH, YEAH. -- FROM A PLANT SPECIES, THERE'S SON EVIDENCE THAT I SOME EVIDENCE IT CAN EXPORT FROM -- THE COFACTOR. SO CAN YOU MODEL -- INTO THIS STRUCTURE YOU HAVE OF THE BACTERIAL? >> WE HAVEN'T TRIED THAT. BUT THAT'S ANOTHER -- WE USED TO WORK ON -- AND COFACTOR ENZYMES AS WELL. THIS IS EVEN MORE PERFECT EXAMPLE. SO NOW THIS REMINDS ME, BUT THERE SEEMS TO BE A KEY SET OF INTERACTIONS THAT REALLY INVOLVE JUST THE INTERACTIONS WITH THE ALPHA AMINO AND CAR BOX ILL GROUP, SO WE FOUND THAT AMINO ACIDS -- WE COULD GET PRETTY STRONG SIMULATION OF ATPASE ACTIVITY WITH AMINO ACIDS, PRESUMABLY BINDING JUST TO WHERE THAT GAMMA GLUTEN WAS BINDING. WE WERE ABLE TO GET A STRUCTURE OF THAT BOUND JUST BECAUSE IT'S SITTING IN ON THAT SIDE. SO I'M A LITTLE EMBARRASSED, WE HAVEN'T TRIED TO SEE IF WE COULD MODEL IN A -- COFACTOR INTERMEDIATE, BUT IT SEEMS LIKE AN IMPORTANT PART FOR THE ACTIVITY IS INVOLVING SORT OF THE NON-SULFUR PART OF THE GLUTATHIONE. >> SO DOUG, I'M FEELING SLIGHTLY BAD FOR YOUR ROBOT IN THE COLD ROOM THERE. SO GIVEN THE NUMBER OF STRUCTURES, AND YOU'VE SHOWN US BEAUTIFUL EXAMPLES THAT HAVE EMERGED FOR WHAT IS CLEARLY A FAMILY WHERE THERE IS A LOT OF STRUCTURAL CONSERVATION, HOW CLOSE ARE YOU NOW WHEN YOU DECIDE TO GO AFTER ONE OF THESE ABC PROTEINS TO BEING ABLE TO MODEL IT COMPUTATIONALLY AND MAKE A PRETTY GOOD PREDICTION ABOUT WHAT THE STRUCTURE WOULD LOOK LIKE? >> WE DON'T HAVE ANY DIRECT EXPERIENCE DOING THIS, SO -- BUT MODELING METHODS ARE BECOMING REALLY QUITE GOOD. I'D SAY ESPECIALLY FOR THE EXPORTERS, YOU KNOW, THAT THERE'S GETTING TO BE MORE AND MORTEMORE TEMPLATES. THE ONE PART WHERE I THINK THEY PROBABLY WOULD HAVE PROBLEMS IS THAT THERE ARE SOME HELICAL IRREGULARITIES THAT ARE IN THE MIDDLE OF THESE, AND SO HOW DEPENDENT THOSE FEATURES ARE ON THE EXACT SEQUENCE, I'D LIKE TO BELIEVE THOSE ARE OFTEN NEAR THE SUBSTRATE BINDING SITES, SO THAT PART MIGHT BE A LITTLE TRICKY, BUT IN GENERAL, I WOULD SAY SORT OF MODELING METHODS, THREADING METHODS ARE BECOMING PRETTY POWERFUL. >> YOU COULD SET UP A CONNEST, JOHN HENRY VERSUS THE MACHINE, SEE WHAT HAPPENS. >> WELL, THERE IS THE STRUCTURE PREDICTION METHOD, AND I WAS INVOLVED -- ACTUALLY IT WAS PRECAST, AND I'D HAVE TO SAY THAT -- THE PREDICTION -- THIS WAS IN THE LATE 80s, '89, WASN'T SO GOOD, WHEREAS NOW THE STRUCTURES, YOU KNOW, EVEN DE NOVO PROTEIN DESIGN AND COMING NEAR THE ORIGINAL TARGET, YOU KNOW, THOSE ARE, I'D SAY, ASTONISHINGLY CLOSE. SO -- >> WELL, VERY GOOD. WE ARE GOING TO -- IF YOU WERE INTERESTED, CONTINUE THE CONVERSATION IN THE LIBRARY WITH OUR SPEAKER OVER COFFEE AND COOKIES. PLEASE JOIN IF YOU CAN, BUT PLEASE ALSO GIVE ONE MORE ROUND OF APPLAUSE TO DR. REES.