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CAPTIONS START IN A FEW MINUTES
SHE IS ALSO A PEER SCHOLAR FOR
MEDICAL SCIENCES.
SHE'S AN EXPERT IN -- RESEARCH
OF ABC TRANPORTERS WHICH ARE
UBIQUITOUS AT EXPORT A VARIETY
OF MOLECULES ACROSS THE CEREBRAL
MEMBRANE.
THESE ARE CRITICAL SURVIVAL
FACTORS BACTERIUM E. COLI FOR
EXAMPLE HAVE 80 DIFFERENT ABC
TRANSPORTERS ON BOARD.
HUMANS HAVE 48 DIFFERENT ABC
TRANSPORTERS -- GENETIC DISEASES
HAVE BEEN LINKED TO THE DEFECTS
IN THESE TRANSPORTERS.
ABC TRANSPORTERS ARE ALSO
CENTRAL TO MULTIDRUG RESISTANCE
IN BACTERIA, FUNGI AND --
TRANSPORTERS ARE A COMPELLING
CLASS OF PROTEIN -- AND FOR
BASIC MEMBRANE BIOLOGY.
DR. CHEN USED THE MODEL
TRANSPORT FROM E. COLI AS A
MODEL SYSTEM TO UNDERSTAND ABC
TRANSPORTERS FOR MORE THAN 30
DECADES SCIENTISTS HAD STACKED
UP DATA THAT GIVE THEM AN
EXCELLENT IDEA OF WHAT THE MODEL
ABC TRANSPORTERS SHOULD BE DOING
YET IT'S DR. CHEN'S WORK THAT
PRODUCED DETAILED PICTURES OF
TRANSPORTERS IN ACTION.
WITH HER SUCCESS IN MODEL
TRANSPORTERS EXTENDING HER
INVESTIGATION INTO ABC EXPORTERS
OF WHICH LESS IS KNOWN ABOUT
THEIR MECHANISM OF FUNCTION SUCH
AS SUBSTRATES -- TO SUBSTRATE
TRANSLOCATION.MORE RECENT HER LAB DETERMIN ED
THE STRUCTURE OF THE MULTIDRUG
TRANSPORTER -- MULTIDRUG
RESISTENT IN CANCER CELLS.
THEY AFFECT ABSORPTION,
DISTRIBUTION AND DRUGS FOUND
RELATED TO CANCERS AND -- TODAY
WE ARE IN FOR A TREAT NATURE'S
MORE INTRICATE MACHINE IN
ACTION.
DR. CHEN WE APPRECIATE YOUR
TAKING THE TIME TO COME HERE TO
DELIVER THE LECTURE.
THE TITLE TALK IS STRUCTURAL AND
MECHANISTIC STUDIES OF ABC
TRANSPORTERS, NATURE'S
FAVORITE -- PLEASE WELCOME OUR
SPEAKER, DR. CHEN.->> THANK YOU FOR THE IN VITATION,
THANK YOU FOR THE WONDERFUL
INTRODUCTION.IT'S REALLY MY HONOR TO BE HE RE
TO GIVE THIS LECTURE.
SO THE VERY NICE INTRODUCTION
WAS ABOUT ABC TRANS PORTERS.
I WOULD LIKE TO POINT OUT
NOWADAYS WE HAVE PROBABLY MORE
THAN 2000 DIFFERENT ABC
TRANSPORTERS IDENTIFIED -- ALL
THE WAY TO HUMANS.
THE FIELD ACTUALLY STARTED IN
THE EARLY 80'S BY THE
REALIZATION THAT TWO PROKARYOTIC
TRANSPORTERS -- SHARE
SIMILARITIES TO A HUMAN MULTI--
WHICH IS FIRST -- HERE AT NIH IN
MICHAEL --'S LAB.
SO THE REALIZATION THOSE GROUP
OF PROTEINS HAVE SPECIAL
SEQUENCE THAT'S ABLE TO BIND AND
HYDROLYZE ATP -- ATP BINDING.
SO NOW WE KNOW ALL ABC
TRANSPORTERS INCLUDING EXPORTERS
AND IMPORTERS ARE COMPOSED OF
TWO TRANSMEMBRANE DOMAINS.
THEY CAN BE *** DIMERS -- THEY
FORM THE PATHWAY FOR DIFFERENT
SUBSTRATES AND ALSO HAVE TWO
COPIES OF INTRACELLULAR OR
CYTOPLASMIC ATP BINDING AND THIS
AGAIN CAN BE *** DIMERS OR
HETERODIMERS.-SO ALL THE MEMBERS OF THE FA MILY
SHOW THIS ARCHITECTURE.
SO THIS IS FOR HISTORICAL
REASONS -- HAS BEEN STARTED FOR
MORE THAN 50 YEARS NOW AS A
MODEL TO UNDERSTAND ABC-TRANSPORTERS.SO TH IS IS A PICTURE TAKEN IN
1981 ALL THE PEOPLE FROM MORE
THAN 70 PEOPLE FROM ALL OVER THE
WORLD COME TOGETHER TO TALK
ABOUT THIS ONE TRANSPORTER.-SO WE CAN FIND A PICTURE FOR P
GLYCOPROTEIN, THIS PICK SURE
WOULD BE EVEN MORE CROWDED.
SO WHAT IS THE TRANSPORTERS ---INTO THE CE LLS TO SUPPORT THE
BACTERIA GROWTH.
IN THE PARAPLASMIC SPACE IT HAS
A BINDING PROTEIN -- BINDING
PROTEIN WILL INTERACT WITH
SUBSTRATE WITH HIGH AFFINITY AND
ON THE CONFIRMATION CHANGE FROM
OPEN TO THE CLOSE CONFIRMATION.
AND IN THE PARAPLASM MEMBRANE,
IT HAS TWO TRANSMEMBRANE CALLED
9 MILD S AND MILD G TOGETHER
WITH ATP SUBUNIT CALLED THE MILD
K.
SO THERE ARE TWO MAJOR EVENTS
HAPPENING IN THE ATP TRANSPORTER-CYCLE.-ON E OF THEM IS THE CHEMICAL
EVENT OF ATP -- THE OTHER IS A
MECHANICAL EVENT OF CONFIRMATION
OR CHANGE.
SO TRANSPORTERS WORK BY WHAT WE
CALL THE OPERATING ACCESS MODEL
WHICH WAS ORIGINALLY PROPOSED IN
1966.THIS IS THE PICTURE FROM VERY
SHORT NATURE.
THIS WILL BRING ACROSS THE
CHEMICAL GRADIENT.
THE INTERNAL BINDING SITES WERE
ONE SIDE OF THE MEMBRANE AND ONE
SIDE OF THE MEMBRANE ONLY.
ARGUMENTING BETWEEN THOSE TWO
CONFIRMATIONS THE -- ALTERING
BETWEEN THOSE TWO
CONFIRMATIONS -- THE MOSTINTERESTING QUEST ION OF COURSE
FOR THE FAMILY IS HOW DOES THE
TWO EVENTS, CHEMICAL EVENT OF
ATP HYDRAULIC AND THE MECHANICAL
EVENT OF CONFIRMATION.
THERE IS BIO CHEMICAL EVENT I
HAVE TO TALK ABOUT FIRST -- WHEN
SHE FIRST RESUBSTITUTE THE MODEL
TRANSPORT INTO -- SO THIS THE
TRANSPORTER HAS A VERY LOW --
THE TITRATING IN THE -- REALLY
DIDN'T DO MUCH.OR YOU JUST ADD IN THE BIND ING
PROTEIN WITHOUT THE SUBSTRATE
YOU HAVE VERY LOW LEVEL
STIMULATION.-SO ONLY WHEN SHE ADD BINDING
PROTEINS TOGETHER WITH THE
MODELS, SHE CAN SEE THE
STIMULATION IS ATP ACTIVITY.
SO THIS IS A VERY IMPORTANT
RESULT BECAUSE IT SHOWS THE
INTERACTIONS OF THE BINDING
PROTEIN IN THE PARAPLASMA SIDE
OF THE MEMBRANE WILL REGULATE
THE ATP ACTIVITY OF THE
TRANSPORTER ON THE OTHER SIDE OF
THE MEMBRANE.SO IT HAS TO BE TRANSFUSED
ACROSS THE MEMBRANE TO REGULATE
ATP ACTIVITY.
NOWADAYS IT'S A COMMON
OBSERVATION NOW ATP ACTIVITY OF
ABC OFTEN REGULATE AT THE
PRESENCE OF SUBVAFT -- SUBSTRATE
AND THIS IS A WAY -- IF THE
SUBSTRATE WAS ABSENT.-WHEN I IF YOU ARE ST ARTED TO
STUDY THE TRANSPORTER IT'S-THINKING MAYBE WE SHOULD
STABILIZE THE TRANSPORTER IN ONE
PARTICULAR CONFIRMATION FOR
CRYSTALLIZATION -- IS A KNOWN
INHIBITOR OF ABC TRANSPORTERS.
SO INDEED WHEN THE TEST -- ON
THE MODEL TRANSPORTER IT
INHIBITS THE ATP HYDROLYSIS AND
THE MOUSE TRANSPORTER.
BUT ALSO THERE'S SOMETHING VERY
INTERESTING I OBSERVED.
SO WHEN WE, THIS IS A SIMPLE
EXPERIMENT -- TO THE C TERMINAL
OF MILD K I CAN PULL DOWN ALL
THREE COMPONENTS OF THE TRANS
PORTER.-SO THIS IS TWO COPIES OF MILD K,
THE ATP COMPONENT AND MILD S IS
ONE OF THE TRANSMEMBRANE
COMPONENTS AND THE MILD G SO
OTHER TRANSMEMBRANE COMPONENTS.
SO THE COMPLEX WITH ATP,
SOMETHING VERY STRANGE HAPPENED.
THOSE ADDITIONAL TURNED OUT TO
BE -- I CAN NEVER GET RID OF.
SO WHAT THIS TELLS US IS THAT
THE TRANSPORTER IN WHAT IS
CALLED THE TRANSLATION STATE FOR
HYDROLYSIS, THE TRANSPORTER
CHANGES TO THE BINDING PROTEIN.-IT BECOME A VERY HIGH AFFINITY
COMPLEX, ALL FOUR COMPONENTS
FORM A VERY STABLE COMPACT.
SO I THOUGHT THIS WOULD BE A
GOOD WAY TO CRYSTALLIZE IT.
SO HOW DO WE -- CATALYTIC
COMPOUND.
THIS IS THE MECHANISM FOR ATP
HYDROLYSIS WHEN THE -- ATP WILL
BIND CATALYTIC BASE WILL
POLARIZE THE WATER AND FORM THE
COVALENT -- UNDER THESE
CONDITIONS ATP WILL BE
HYDROLYZED -- INTERMEDIATE THERE
ARE SEVERAL WAYS WE CAN DO.
ONE IS WE CAN MAKE A MUTATION
AND A FORM OF THIS BASE.
SO WITH THIS MUTATION ATP WILL
BIND WITH HIGH AFFINITY BUT NOT
BEING ABLE TO HYDROLYZE IT --
WILL MIMIC THE COVALENT COMPLEX
OF THE ATP DOING HYDROLYSIS AND
ATTRACTS THE COMPLEX.
SO THE MULTIPLE WAYS AND -- TO
CAP THE TRANSITION STATE AT
LEAST ONE STRUCTURE WHICH IS
SHOWN HERE.
SO HERE IS THE MEMBRANE
COMPONENT OF THE TRANSPORTER
BLUE AND YELLOW ARE THE TWO
TRANSMEMBRANE SUBUNITS MILD S
AND THE MILD G.
PURPLE HERE IS THE BINDING
PROTEIN AND ON THE OTHER SIDE OF
THE MEMBRANE THE GREEN AND THE
RED ARE THE TWO ATP COMPONENT OF
MILD K.
AND MOST INTERESTING THING WE
WANT TO FOCUS ON ABOUT THIS
COMPLEX IS THE TRANSPORTER
SUBSTRAIGHT AND LIGAND ATP.
SO FIRST LET'S LOOK AT THE ATP.
SO IN THIS COMPLEX, TWO ATP'S
ARE TRAPPED AT AN INTERFACE
BETWEEN THE TWO MILD K SUBUNITS
SO WE CALL THIS A CLOSED MILD K.
A CLOSED DIMER WITH TWO ATP'S
RIGHT AT INTERFACE
SIMULTANEOUSLY INTERACTING WITH
THE BOTH SUBUNITS.
SO A DETAILED MAP VIEW OF THIS
CONFIRMATION ATP IS A SANDWICH
WHEN THE WALKER A WALKER B MOTIF
OF ONE SUBUNIT AND -- FOR THE
SIGNATURE MOTIF FOR ABC
TRANSPORTER SINCE THEY ARE
HIGHLY CONSERVING ABC
TRANSPORTERS.
SO ATP SIMULTANEOUSLY, THEY BIN
INTO BOTH SIDES OF THE DIMER AND
THE PURPOSE IS FOR HYDROLYSIS.
SO IN THIS CONVERSATION ATP IS
READY TO BE HYDROLYZED.
WHERE IS THE SUBSTRATE.
IT'S SUPPOSED TO BE DELIVERED BY
THE MOUSE'S BINDING PROTEIN AND
THROUGH THE WORK OF -- MY POST
DOC MENTOR WE KNOW THAT MILD
BINDING HAS TWO MAJOR
CONFIRMATIONS.
THEY FORM A CLOSE CONFIRMATION
WHERE MODEL IS BUBBLE BETWEEN
THE TWO LOBES OF MBP.
IN THE ABSENCE OF THE SUBSTRATE
THE TWO LOBES ARE IN THE MORE
OPEN CONFIRMATION.
SO WE'LL CALL THIS THE OPEN
CONFIRMATION AND THE BINDING
WITH THE SUBSTRATE WILL BE THE
CLOSE CONFIRMATION.
SO IN THESE COMPLEX THE BINDING
PROTEIN -- IS TRAPPED IN THE
OPEN CONFIRMATION AND WHERE IS
THE SUBSTRATE.
THE SUBSTRATE NO LONGER BOUND
WITH MPT.
INSTEAD IT'S DELIVERED INTO THE
TRANSMEMBRANE SUBUNIT AND HERE'S
THE DENSITY FOR THE MODEL
TOGETHER WITH SEVERAL WATER
MOLECULES MEDIATING INTERACTION-BETWEEN TH E SUBSTRATE AND THE
TRANSMEMBRANE PROTEIN.
AND HERE ALSO A POCKET FORMED
INTERFACE WHEN MILD S MILD G AND
THE BINDING PROTEIN.
SO THIS IS LARGE ENOUGH --
GLUCOSE AND IT IS OCCLUDED FROM
THE MEMBRANE AND FROM THE
PARAPLASMA SPACE SO MOUSE WILL
NOT BE ABLE TO ESCAPE BACK.
BY THIS WAY IN THIS CONFIRMATION
BASICALLY THE PROTEIN HALF OF
THE SUBSTRATE INSIDE THE
MEMBRANE IT HAS NO WHERE TO GO
BACK.
AND WE THINK OF THIS IS
IMPORTANT TO MAKE SURE MOUSE
WILL GO TRANSPORTED IN ONE
DIRECTION INSTEAD OF ESCAPING
BACK TO THE PARAPLASMA SPACE.
SO WHERE DOES THIS STRUCTURE
REPRESENT IN THE CONTEXT OF
ACCESS MODEL.
THIS IS THE FLAT VIEW.
WE JUST TAKE -- ACROSS THE
PROTEIN AND NOW YOU CAN CLEARLY
SEE THE BINDING POCKET.
THIS POCKET IS OPEN TO THE
PARAPLASMIC SIDE BY THE BINDING
PROTEIN.
SO WE INFILTRATED THIS STRUCTURE
OUTWARD FACING CONFIRMATION
WHERE THE SUBSTRATE IS BINDING
SITE IS FACING OUTSIDE FACING
THE PARAPLASMA SPACE.
SO HERE INSTEAD OF OPEN TO THE
PARAPLASMIC SPACE IS ACTUALLY
CAPPED BY THE BINDING PROTEIN.
AND THIS BY THE BINDING PROTEIN
IS IMPORTANT BECAUSE WE KNOW THE
AFFINITY OF THE BINDING SITE IS
QUITE LOW.SO THIS WILL BE IMPORTANT TO
MAKE SURE THE SUBSTRATE WILL NOT
ESCAPE BACK.
SO THE ORDER CONFIRMATION, THE
INWARD FACING CONFIRMATION WE
WERE ABLE TO OBTAIN THE CRYSTALS
IN THE ABSENCE OF BINDING
PROTEINS AND ANY NUCLEOTIDE.SO THIS IS THE LOW RESOLUTION
STRUCTURE BUT NEVERTHELESS YOU
CAN SEE THE BASIC ARCHITECTURE.
THE MOUSE BINDING SITE NOW IS
FACING INSIDE THE CELL CORRECTED
TO THE CYTOPLASM AND THE TWO
MILD K SUBUNITS NO LONGER
CONTACT EACH OTHER SO IT FORMS
WHAT WE CALL THE OPEN DIMER.
SO WHEN WE PUT THOSE TWO
STRUCTURES NEXT TO EACH OTHER,
WE CAN BASICALLY CAPTURE THE TWO
BASIC CONFIRMATIONS IN A
TRANSPORTER CYCLE.
INWARD FACING CONFIRMATION WITH
THE TWO NUCLEOTIDE BINDING
DOMAINS OPEN, SEPARATED FROMEACH OTHER AND THE BINDING SITE
IS FACING INSIDE THE CELL.
AND OUR FACING CONFIRMATION
WHERE THE MOUSE BINDING SITE IS
FACING MBP AND THE TWO ATP
SANDWICH WHAT WE CALL THE CLOSE
DIMER OF MILD K.
IT TURNS OUT THE TRANSITION
BETWEEN THE INWARD FACING
CONFIRMATION TO THE OUTWARD
FACING CONFIRMATION REQUIRES NOT
ONLY ATP BUT ALSO THE BINDING
PROTEIN.
SO THIS IS -- AMY DAVIS' LAB
WHEN SHE WANTS TO UNDERSTAND
WHAT DOES THE BINDING PROTEIN
REALLY DO TO STIMULATE ATP
HYDROLYSIS.
WHAT SHE HAS DONE, SHE PUT A
STIMULATOR AT THE TWO MBD'S
INSIDE THE CELL ON MILD K AND --
INTERACTIONS OF THOSE TWO -- SHE
CAN DISTINGUISH INWARD FACING
OPEN DIMER WITH THE OUTWARD
FACING CLOSER DIMER.BASICALLY WHAT HER DAT A SHOWS IS
IF YOU TAKE THE -- INWARD FACING
TRANSPORTER ADDING ATP, IT DOES
NOT CAUSE ANY CONFIRMATIONAL-CHANGE.
ONLY IN THE PRESENCE WHEN BOTH
BINDING PROTEIN AND ATP INFORMED
OUTWARD FACING CONFIRMATION --
WITH THE TRANSPORTER IS GOING TO
DO SOMETHING WITH THE
TRANSPORTER.WE TRY TO UNDERSTAND BY
BASICALLY DETERMINE THE
STRUCTURE OF WHAT WE CALL THE
PRETRANSLOCATION STATE.
SO THIS STRUCTURE IS IN THE
PRESENCE OF BINDING PROTEIN WITH
HIGH CONCENTRATIONS OF SUGAR AND
ALSO IN THE ABSENCE OF A
NUCLEOTIDE NO ATP OR ADP.
SO WE CALL THIS THE
PRETRANSLOCATION STATE.YOU CAN SEE THE BIE BLEDDING
PROTEIN IN THE CLOSER
CONFIRMATION WITH THE SUBSTRATE
STILL BINDS TO IT.
AND WE CAN ALSO SEE THE BINDING
SITE INSIDE THE MEMBRANE BECAUSE
WE HAD HIGH CONCENTRATIONS OF --
SO WE SEE BOUND HERE.
THIS STRUCTURE REPRESENTS THE
PRETRANSLOCATION BEFORE -- HASBEEN DELIVER ED TO THIS SITE.
BECAUSE THIS BINDING SITE HAS
MUCH HIGHER AFFINITY IN
COMPARISON TO THE TRANSMEMBRANE
BINDING SITE.
SO WITH ALL THESE STRUCTURES,
WHAT CAN WE LEARN ABOUT THE
SYSTEM.
WHAT KIND OF MECHANISTIC
QUESTIONS CAN WE ADDRESS.
SO FIRST WE'D LIKE TO SEE THE
STRUCTURED DETAILS OF
ALTERNATING ACCESS MODELS.
HERE WE PRESENT THE THREE
STRUCTURES.
INWARD FACING STRUCTURE, WE ALSO
BELIEVE IT REPRESENTS WHAT WE
CALL THE RESTING STATE BECAUSE
IT HAS VERY LOW ATP ACTIVITY AND
THE SUBSTRAIGHT IS ABSENT.
NOW WE ALSO PRESENT OUTWARD
FACING STRUCTURE CONFIRMATION
WHERE MILD MOUSE IS ALREADY
DELIVERED FROM THE BINDING SITE
FROM MDP INTO THE MEMBRANE AND
THE TWO ATP'S ARE BEING
POSITIONED AT THE DIMER
INTERFACE READY TO BE
HYDROLYZED.AND PRESENT AN INTERMEDIATE
BETWEEN THE INWARD FACING AND
OUTWARD FACING STATE WE CALL THE
PRETRANSLOCATION STATE WHICH
REPRESENTS THE INITIAL CONTACT
OF THE BINDING PROTEIN WITH THE
TRANSPORT ARE.
WHEN WE ANALYZE THE STRUCK
CHOOSH BETWEEN THOSE
CONFIRMATIONS THEY ARE SMALL
LOCAL CONFIRMATIONAL CHANGES.
THE CONFIRMATIONAL CHANGE CAN
DESCRIBE AS RIGID BODY LOCATIONS
OF THE TWO TRANSMEMBRANE DOMAINS
AND THE TWO NUCLEOTIDE BINDING
DOMAINS.I SHOULD POINT OUT MILD K IS --
EACH HAS TWO SUBDOMAINS --
NUCLEOTIDE BINDING DOMAIN WILL
HYDROLYZE ATM AND THE C TERMINAL
REGULAR TO THE DOMAIN
INTERACTING WITH OTHER
REGULATORY PROTEINS.
SO IN DOING THIS TRANSITION, THE
TWO NUCLEOTIDE BINDING DOMAINS
WILL ROTATE INWARDS TO EACH
OTHER AND THE TWO MBD'S, THE TWO
TRANSMEMBRANES WILL ALSO ROTATE.
SO THIS IS THE TRANSITION
BETWEEN THE INWARD FACING REST
STATE -- INDUCE THE BINDING
PROTEIN.
AND THIS IS THE TRANSITION
BETWEEN THE PRETRANSLOCATION
STATE -- BIND HERE TOWARDS THE
OUTWARD FACING STATE WHERE IT IS
DELIVERED INTO THE MEMBRANE.-THEN YOU CAN SEE THE TWO LOBES
OF MDP OPEN UP TO RELEASE MODELS
AND THE TWO TRANSMEMBRANE
DOMAINS WILL ROTATE TO RECEIVE
THE MODEL AND THE TWO MBP WILL
ROTATE INWARD SO THEY CAN BIND
AND HYDROLYZE ATP.
WHEN YOU LOOK AT THIS MOVIE
PRETTY MUCH ALL PROTEIN PROTEIN
INTERFACE ARE CHANGING EXCEPT
FOR TWO PLACES.
ONE PLACE IS AT THE C TERMINAL.
THE REGULATORY DOMAIN INTERFACE
OF MILD K DIMER IS MAINTAINED IN
THIS TRANSITION.
AND THE OTHER INTERFACE IS
ACROSS WHAT WE CALL THE PG.
THIS IS THE LARGE PARAPLASMIC
LOOP FROM THE MILD S SUBUNIT
WITH MBP.
SO THIS INTERFACE IS MAINTAINED
AND THIS INTERFACE IS
MAINTAINED.
WE LIKE TO TEST WHETHER THIS
GRAPHIC OBSERVED CONFIRMATION
CHANGE IS -- DURING A
TRANSPORTER CYCLE.AGAIN THIS IS A WORK FRO M AMY
DAVIDSON.-WHAT SHE DID IS SHE DECIDED TO
CROSS LINK THE REGULATORY DOMAIN
WHICH WE KNOW WILL MAINTAIN ITS
INTERFACE DURING THE TRANSPORTER
CYCLE BY 215.
SO UNDER OXIDATION CONDITIONS-THE DIMER IS ALMOST 100% OF MILD
K FORM OF DIMERS.
SO THOSE TWO DOMAIN ARE CROSS
LINKED AND THE CROSS LINK
TRANSPORTER WORKS JUST FINE.
SO THIS MEANS IN THE TRANSPORTER
CYCLE -- THE TWO REGULATORY
DOMAINS DO NOT NEED TO
DEASSOCIATE JUST AS WE SEE IN
THE CRYSTAL STRUCTURES.
WE ALSO -- THE INTERFACE BETWEEN
THE P2 DOMAIN AND THE MBP WHICH
IS IN THE PARAPLASMIC SPACE.
BY DOING ALSO CROSS LINKING WE
CAN CROSS LINK MDP WITH MILD S A
HUNDRED PERCENT.
THIS CROSS LINK TRANSPORTER
BETWEEN FUNCTION JUST FINE.
SO IN CONTRAST THE CROSS LINK AT
THE PROTEAN PROTEIN INTERFACE
THAT'S SUPPOSED TO CHANGE.
WE SEE SOMETHING TOTALLY
DIFFERENT.
SO ONE EXAMPLE IS YOU CAN SEE
230 -- COMING TOGETHER FOR WHAT
WE BELIEVE FORM THE PARAPLALS MA
IN ASMA
IN THE INWARD FACING
CONFIRMATION.
AND WE CAN CROSS LINK THIS
TRANSPORTER WILL SPONTANEOUSLY
CROSS LINK THE MILD S AND THE
MILD G WILL BECOME ONE BEND BE
CROSS LINK.
SO THE CROSS LINK STANDS FOR THE
K NO LONGER FUNCTION BECAUSE THE
TRANSITION FROM THE INWARD
FACING STRUCTURE TO THE
PRETRANSLOCATION STRUCTURE THEN
FURTHER TOWARDS THE OUTWARD
FATING STRUCTURE WILL REQUIRE
THE TWO SMALL MOLECULES THE TWO
AMINO ACIDS TO BE SEPARATED SO
THEY OPEN UP THE -- SO THE MOUSE
CAN DELIVER HERE.
SO UNDER OXIDATION CONDITIONS,THE CROSS L INK TRANSPORTER NO
LONGER FUNCTIONS AND PUTS THE --
BIOCHEMICAL OR EXPERIMENT WAS
DONE TO VALIDATE THE
CONFIRMATIONAL CHANGES WE
OBSERVE IN THE CRYSTAL
STRUCTURE.
THE NEXT QUESTION I HAVE TO
ADDRESS I MENTIONED FOR ATP
TRANSPORTERS IT'S VERY COMMONTHAT THE PRES ENCE OF THE
SUBSTRATE WILL TURN ON THE ATP'S
ACTIVITY.
AND WHEN THE BINDING PROTEIN
DEPENDS SUCH AS TRANSPORTER IT'S
THE PRESENCE OF THE MOUSE
BINDING PROTEIN TOGETHER WITH
THE MODEL WILL STIMULATE ATP'S
ACTIVITY.
SO -- BY COMPARING THE STRUCTURE
OF THE RESTING STATE THE
TRANSPORTER IN THE ABSENCE OF
ANY SUBSTRATE AND TOGETHER WITH
THE TRANSPORTER THAT'S BEEN
STABILIZED BY THE BINDING
PROTEIN WE CAN SEE THE
INTERACTION OF THE BINDING
PROTEIN WILL CROSS GLOBAL
CONFIRMATIONAL CHANGE OFF OF THE
TRANSPORTER.
YOU NEED THIS PQ DOMAIN WHICH IS
ABSENT IN THE INWARD FACING
STRUCTURE BECOME ORGANIZED AND
INTERACT WITH THE BINDING
PROTEIN.
WE BELIEVE IN THIS CONFIRMATION
THE P2 DOMAIN IS QUITE FLEXIBLE.
IT'S MOVING AROUND LIKE A
RECEPTOR.
ONCE THE BINDING PROTEIN WILL-HELP TO BRIN G THE BINDING
PROTEIN TOWARDS THE TRANSPORTER
TO FORM THIS INITIAL COMPLEX.
THE MORE IMPORTANTLY THE
INTERACTION OF THE BINDING
PROTEIN WITH THE TRANSPORTERWILL CROSS ROT ATION OF THE
TRANSMEMBRANE DOMAIN.
AND YOU CAN SEE THAT BY
COMPARING THE TRANSMEMBRANE
BINDING SITE IN THE INWARD
FACING STRUCTURE WITHOUT THE BIN
BINDING PROTEIN NOW WE CHANGE
FROM INWARD FACING BINDING -- SO
THE TWO SUBUNITS ROTATED
RELATIVELY TOWARD EACH OTHER AND
CLOSE THESE ENDS.
THESE ROTATIONS WILL BRING THE
TO BINDING DOMAINS ON TO THOSE
TWO SUBUNITS CLOSE TO EACH
OTHER.
AND IT WILL BE CLOSE ENOUGH.
NOW ATP BINDING WILL COMPLETE
THE TRANSITION TO THE OUTWARD
FACING STATE.
SO THIS IS A CLOSE UP VIEW OF
THE CONFIRMATION REGARDING THE
ATP BINDING SITE.
SO THIS IS THE WALKER A MOTIF
AND 192 THAT'S WE KNOW IN FACT
WAS ATP.
SO IN THE ABSENCE OF ANY BINDING
PROTEINS IN THE OPEN DIMER
CONFIRMATION OF MILD K, THE
WALKER A MOTIF AND ATP BINDING
MOTIF IS NOT IN CONTACT WITH ANY
RESIDUE FROM THE OTHER SIDE OF
THE, IN THE OTHER SUBUNITS SO
THEY ARE SEPARATE FROM EACH
OTHER.-BUT THE INTERACTION OF THE
BINDING PROTEIN WITH THE
TRANSPORTER WILL CAUSE THE MBD
TO ROTATE INWARD.
WITH THOSE CONFIGURATION THOSE
RUSE DUES IS MAKING HYDROGEN
BONDING WITH THE SECOND MBD.
SO NOW THE TWO MBD START TO
COMMUNICATE WITH EACH OTHER.
AND IN THIS CONFIGURATION ATP
COMES OVER WILL FURTHER CAUSE
CLOSURE OF THIS MILD K
INTERFACE.-NOW WE FORM THE CLOSE MILD K
WHERE ATP'S POSITION AND
SIMULTANEOUSLY INTERACTING WITHTHE SUBUNIT .
SO IN THIS CLOSE CONFIRMATIONATP AS I MENT IONED BEFORE NOW IS
THE POSITION ALL THE CAT
POLITICAL RESIDUES ARE IN THE
RIGHT PLACE TO HYDROLYZE ATP.-ONLY IN THE CLOSE THE
CONFIRMATION ATP IS HYDROLYZED.
SO FOR THIS OUTWARD FACING STATE
THE MOLECULES THE TWO THINGS --
FROM THE BINDING PROTEIN INTO
THE MEMBRANE THE SECOND THE
THING IS IT WILL PLACE ATP IN A
POSITION TO BE HYDROLYZED.
SO THE REASON ONLY THE CLOSER
DIMER CAN HYDROLYZE ATP ANALOGY
TO WHAT WE KNOW -- DISCOVERED IN
MANY OTHER ATP.
THIS IS AN EXAMPLE FOR THE S1
ATPASE .รบ
THIS IS THE WALKER A MOTIF OF
ONE SUBUNIT THAT WILL BIND ATP
AND IT HAS ALL THE CAT POLITICAL
RHESUS DOOTZ ABLE TO HYDROLYZE
ATP.HOWEVER IT IS NOT ABLE TO
HYDROLYZE ATP IN THE ABSENCE
OF -- WHICH WILL BE SUPPLIED BY
ANOTHER SUBUNIT.-SO THE FUNCTION OF THE AR GININE
FINGER IS TO POSITION THE
PHOSPHATE SO IT WILL BE ABLE TO
DEHYDROLYZE.-SO HERE IN THE MODEL
TRANSPORTER -- CAN BE REPLACED-WHAT WE CAL L THE SIGNATURE PHASE
OF THE ATP TRANSPORTERS -- IT-DOES EXACTLY THE SAME THING AS
AVERAGE MEAN FINGER.
IT POSITIONS THE PHOSPHATE TO BE
HYDROLYZED.AND AFTER THIS HYPOTHESES BY
EXPERIMENT KIND OF -- MORE OR
LESS FOR ACCIDENTS.
WE TOOK ADVANTAGE OF WE INFORMED
THE PRETRANSLOCATION STAGE BY
TWO METHODS.
ONE WAY IS CO-CRYSTALLIZE
BINDING PROTEIN WILD TYPE
BINDING PROTEIN WITH HIGH
CONCENTRATIONS OF MODEL.
ANOTHER WAY WE TOOK APPROACH IS
WE PUT -- CROSS LINK AND
STABILIZE THE BINDING PROTEIN IN
THE CONFIRMATION.-SO THEY CAN UNLOCK THE
TRANSPORTER IN THISPRETRANSLOCATION STAGE WHERE THE
TWO MBD'S ARE SEPARATED FROM
EACH OTHER.
SO WE SAY OKAY LET'S NOW ATP AND
SEE IF THERE'S ONLY LOCAL
CONFIRMATION OF CHANGE ACROSS
THE ATP BINDING.THAT WAS THE ORIGINAL DOSE FOR
THIS EXPERIMENT.
SO THE RESULTS CAME OUT -- SO IN
THE CROSS LINK MBP ONE WOULD
PREVENT MBP FROM OPENING.
THIS IS A HYDROLYZABLE ATP
ANALOGUE.-WHAT WE SEE IS WE CAN SEE THE
CRYSTAL STAY IN THE SAME
CONFIRMATION AND WE CAN SEE
BINDING TO THE NUCLEAR BINDING
SITE.BUT EQUALLY USED THE WILD TYPE
MBP -- WE WERE VERY SURPRISED TO
SEE THE -- OPENED UP AND THE
WHOLE TRANSPORTER TRANSITION TO
THE OUTWARD FACING CONFIRMATION.
AND THE FIRST THING WITH FIRST
TIME WE DID THIS WAS -- SO WE
RADIATED THREE TIMES --
EXPERIMENT AND AGAIN EVERY TIME
SINGLE TIME WAS TO HAVING THOSE
OUTWARD FACING CONFIRMATIONS.
SO WE HAD A VERY SPECIAL
ARRANGEMENT IN OUR CRYSTALS AND
THIS CONFIRMATIONAL SOMEHOW
DESTROY IT.
BUT WE ALSO SEE SOMETHING VERY
INTERESTING.
SO IN THIS OUTWARD FACING
CONFIRMATION WE CAN SEE THE --
SO THIS EXPERIMENT TELLS US, IN
THIS EXPERIMENT -- DO NOT GET
HYDROLYZED.
BUT WE LOOK AT THE DENSITIES FOR
THE OPEN WHEN THE MBP'S ARE
OPERATED FROM EACH OTHER.
WE CAN ONLY SEE A -- WHEN WE
LOOK AT THIS STRUCTURE WE SAY
UH-HUH.THE REASON WE DON'T SEE THE --
GAMMA PHOSPHATE IS BECAUSE THE
GAMMA PHOSPHATE IS NOT IN ONE
PLACE IT'S PROBABLY MOVINGAROUND.-THAT'S W HY IT HAPPENS NO
DENSITY.-AND THE REASON THE GAMMA
PHOSPHATE IS MOVING AROUND IS
BECAUSE -- IS NOT IN PLACE TO
POSITION IT.AND WE WENT TO THE LITERATURE TO
COMPARE.THERE ARE MANY HIGH RESOLUTION
STRUCTURES THAT WAS ISOLATED MBD
WITH ATP BOUND.SO WE TOOK A POSITION THOSE
STRUCTURES THE POSITION FOR ACP
IS VERY WELL BUT THE POSITION
FOR THE GAMMA PHOSPHATE IS VERY
DIFFERENT CRISES IN SOME
STRUCTURES.SO THIS ACTUALLY IN FORCE ONLY
IN THE CLOSE CONFIRMATION, CLOSE
DIEMPLE CONFIRMATIONS THE
PRESENCE OF -- WILL POSITION THE
GAMMA PHOSPHATES FOR HYDROLYSIS.
SO THIS AGAIN EXPLAINS WHY THE
OPEN MBD DIMER HAS VERY LOW ATPACTIVITY.
DURING ATP HYDROLYSIS THE
CONFIRMATION OF ATP ACTUALLY
CHANGES.
SO IN THE GRAND STATE THE GOT MA
PHOSPHATE FORMS THE --
CONFIRMATION AND -- WHAT WE CALL
THE -- BASICALLY THOSE FOUR
ATOMS PHOSPHATE AND THE THREE --
FROM ONE PLANE.-ON EACH SIDE WE HAVE OXYGE N.
ONE IS THE OXYGEN FROM ADP.
THE OTHER ONE IS ATTACKING THE
OXYGENS ON THE ATTACKING WATER.
THIS IS A VERY SPECIAL STRUCTURE
AND THE TRANSITION STATE FOR ATP
HYDROLYSIS.
SO IN THE FIELD PEOPLE WONDER
WHETHER THE TRANSITION FROM THE
TETRAHEDRAL CONFIRMATION TO THE
TRANSITION STATE WILL CROSS ANY
GLOBAL CONFIRMATION CHANGE FOR
THE PROTEIN.
SO HERE WE ADDRESS THAT QUESTION
BY BASICALLY CRYSTALLIZE
DETERMINE THE HIGH RESOLUTION
STRUCTURE OF THE TRANSPORTER IN
CONTRAST WITH THE NUCLEOTIDES --
OR THE TRANSITION STATE.
SO HERE IS THE DENSITY FOR THE
GROUND STATE, THIS IS DETERMINED
BY THE AMP -- YOU CAN SEE THE
NICE TETRAHEDRAL STRUCTURE OF
THE GAMMA PHOSPHATE IN THIS
CONFIRMATION.
WHEN WE HAVE THE TRANSITION
STATE WITH ALUMINUM FLUORIDE --
WE CAN SEE THE CONFIRMATION.
IT'S A GLOBAL CONFIRMATION FOR
THE STRUCTURE DIDN'T CHANGE AND
BUT NOW YOU CAN SEE THE NICE --
FOR THE GAMMA PHOSPHATE AND THE
POSITION TO ATTACK A PHOSPHATE.
SO THIS IS BASICALLY TELLS US
FOR ABC TRANSPORTERS THE
FORMATION OF THE TRANSITION
STATE OF THE HYDROLYSIS DID NOT
CROSS ANY GLOBAL CONFIRMATION OR
CHANGE FOR THE PROTEIN.
IT'S AN AREA CHEMICAL FORHYDROLYSIS.
SO THE NEXT QUESTION WE LIKE TO
AWE DRESS IS HOW THE ATP
HYDROLYSIS ENABLES SUBSTRATE
TRANSPORTER.
SO MOST OF COURSE THE
STRAIGHTFORWARD WAYS WOULD LIKE
TO DETERMINE THE STRUCTURE OF
THE POST HYDROLYSIS STATE TO SEE
WHAT HAPPENS TO -- AFTER
HYDROLYSIS WHICH WE WERE NOT
SUCCESSFUL.AND I BELIEVE THERE'S A REASON.
THE REASON IS IF YOU LOOK AT THE
STRUCTURE OF THE OUTWARD FACING
STATE, THE DIMER, THE OPEN
CLOSED DIMER IS BASICALLY LINKED
TOGETHER FOR THE GAMMA
PHOSPHATE.-AND SO YOU CAN IMAGINE AFTER AT P
HYDROLYSIS, THIS BOND IS GOING
TO BREAK AND THEN AFTER RELEASE
OF THE INORGANIC PHOSPHATES,
THIS DIMER IS NO LONGER TO BE
STABLE.
SO IT WILL OPEN UP -- PROBABLY
WILL BE VERY SIMILAR TO THE
PRETRANSLOCATION STAGE.-WHEN YOU OPENED UP THE MBD ARE
DIMER YOU WILL CROSS ROTATION OF
THE TRANSMEMBRANE DOMAIN.
BUT THERE YOU WERE FORCE THE
MDB'S TO CLOSE.
BUT THIS TIME THE SUBSTRATE
MODEL IS ALREADY DELIVERED TO
THE MEMBRANE SITE.
SO MBP WILL HAVE TO CLOSE IN THE
ABSENCE OF THE SUBSTRATE WHICH
WE KNOW IS VERY IS UNFAVORABLE.
SO IN THIS CASE I THINK OF THIS
COMPLEX WILL QUICKLY RELAX INTO
THE INWARD FACING CONFIRMATION
MBT WILL DEASSOCIATE AND FIND
ANOTHER MOUSE TO INTERACT WITH.
AND FROM STUDIES OF EPR AND
ISOLATED MBD WE KNOW AFTER ATP
HYDROLYSIS THE TWO MBD'S WILL
OPEN UP.BASED ON THOSE EVIDENCE WE
PROPOSE THIS IS THE BASIC
HYDROLYSIS CYCLE.
THE ATP BINDING WILL INDUCE
OUTWARD FACING STAGE.
DELIVER INTO THE MEMBRANE AND
ATP WILL BE READY TO
DEHYDROLYZE.
THEN THE ENERGY FROM ATP
HYDROLYSIS WILL COMPLETE THE
CYCLE ALL THE WAY BACK TO THE
INWARD FACING STATE OR THE
SUBSTRATE WILL BE DEASSOCIATED
INTO THE MEMBRANE INSIDE THECELL.
SO THIS IS OUR UNDERSTANDING OF
THE TRANSPORTER CYCLE.-AND SO ONCE WE KNOW HOW THE ABC
TRANSPORTER WORKS THE NATURAL
QUESTION IS CAN WE REGULATE
THESE ACTIVITIES.
IT TURNS OUT THAT NATURE HAS
ALREADY COME UP WITH A SOLUTIONFOR THE E. COLI MOUSE
TRANSPORTER.
SO THIS IS RELATED TO WHAT WE
KNOW AS CARBON CATABOLIC
REPRESSION.IT TURNS OUT -- IS NOT E. COLI
FAVORITE SUGAR.
SO FROM THE ENERGETIC POINT OF
VIEW, THERE ARE CERTAIN
CARBOHYDRATES WERE PREFERRED BY
E. COLI BECAUSE IT WILL BE
QUICKLY METABOLIZED.
SO IN THE PRESENCE OF THOSE
CARBOHYDRATES WHAT E. COLI WILL
DO IS PRESS EXPRESSION AND THE
TRANSPORTER ACTIVITIES OF ALL
OTHER CARBOHYDRATES.-AND THIS IS A NOMINAL NOW
REGULATED ABOUT FIVE TO
TEN PERCENT OCCUR LIE GENES.
THIS REGULATION IS BEING
ACHIEVED THROUGH ONE PROTEIN
CALLED QA.
THE ENZYMES HERE OF THE GLUCOSE
TRANSPORTER PART OF THE PTS
SYSTEM WHICH IS STARTED HERE AT
NIH.
SO IN THE ABSENCE OF A PREFERRED
SUGAR AS GLUCOSE, ETA WILL
MOSTLY IN THE PHOSPHORYLATED
STATE.
THE TOAST FORELATED EQA WILL
TURN AROUND CYCLIC A AND P
SYNTHESIS WHICH IN TURN WILL
TURN AROUND.
A LOT -- WHICH I'LL ENCODE --
OTHER CARBON SOURCE.
WHEN GLUCOSE IS PRESENT, IT WILL
BE TRANSPORTED INTO THE SYSTEM
THROUGH THE PTS SYSTEM AND
PHOSPHORYLATED AND IT ORIGINATED
FROM PEP THROUGH A CAST
INDICATED OF REACTIONS.
AND THE INTERMEDIATE OF THIS
REACTION NOW E6789 TA WILL BE
SHIFTED UPON THE PHOSPHORYLATED
TO DEPHOSPHORYLATED STATE.
NOW THE DEPHOSPHORYLATED ETA
WILL GO AHEAD AND THE BLOCK OF
ACTIVITIES OFF A NUMBER OF SUGAR
TRANSPORTERS.
WHICH INCLUDES THE LACK OF Y
TRANSPORTER WILL PICK UP
LACTOSE -- BY INTERACTING
WITH -- SO THE QUESTION IS HOW
DOES E2A DO IT.
OKAY.-ONE MORE THING I WANT TO POINT
OUT.
PHYSICALLY BLOCKING THE
ACTIVITIES OF THOSETRANSPORTERS, ACTUALLY THEY ARE
TWO THINGS.
ONE IS PREVENT IMMEDIATE UPTAKE
OFF THESE SUGARS.
ANOTHER THING IS ACTUALLY WILL
DOWN REGULATE THE EXPRESSION OF
ALL THE GENES THAT WILL ENABLE
TRANSPORTER METABOLISM OF THOSE
SUGAR.BOAST THOSE CARBOHYDRATES TURNSOUT T O BE THE INDUCERS OF THIS.
IT'S BEST TO STUDY THE SYSTEM WE
ALL LEARN IT'S THE LACTOSE, THE
LACK OF IN THE ABSENCE OF ANY
LACTOSE E COLI DOES NOT WASTE
ENERGY TO MAKE ENZYME THAT WILL
BREAK DOWN LACTOSE.-SO WHAT HAPPENS THERE' S A
REPRESSER WILL BIND TO THE
OPERATOR WILL PREVENT-EXPRESSION -- WILL B E
TRANSPORTED INTO THE CELL.IS ICE FOAFERL O F LACTOSE WILL
INTERACT WITH THE REPRESSER AND
THIS INTERACTION WILL BLOCK THE
BINDING OF REPRESSER TO THE
OPERATOR.-NOW POLYMERASE WILL BIND AND.
GO AHEAD AND MAKE A GENE WILL
ENABLE LACTOSE UP TAKE AND THE
BREAK DOWN.
SO MILD -- ACTIVATOR WE'LL
CALLED THE MILD T.
SO ACTIVATION OF THE MILD -- ALL
THE GENE THAT WILL ENABLE
TRANSPORT AND BREAK DOWN IS
UNDER REGULATION OF MILD T BUT
ALSO REQUIRED THE PRESENCE OF --
SO WHEN ETA PREVENTS TRANSPORTER
IT WILL ALSO DOWN REGULATE THE
EXPRESSION OF ALL THE MILD -- SO
E2A IS AN EVENTUAL MOLECULE IN
THE SYSTEM HAS BEEN STARTED FORMANY YEARS THROUGH MANY
LABORATORIES.THROUGH THE WORK WE KNOW E2A IS
A MOLECULE WITH 168 MOLECULES --
FLEXIBLE AGENTS AND MAJOR
STRUCTURE WHICH LOOKS LIKE THIS.
SO WE KNOW PHOSPHORYLATION OR
INTERACTIONS WITH OTHER
MOLECULES WILL CHANGE THE
CONFIRMATION OF E2A.
THE STRUCTURE OF E2A WAS DOWN
STREAM -- AS WELL AS KINASE
DETERMINE AND THEY SHOW ACTUALLY
E2A USE A COMMON SOURCES
INTERACTING WITH ALL OF THE
MOLECULES.AND THE ONE -- WHICH IS YELLOW
HERE.
ALL OF THE INTERFACE INVOLVES
HISTIDINE 90 WHICH IS KNOWN AS
PHOSPHORYLATION SIDE FOR E2A.
SO WE LIKE TO KNOW HOW E2A IN
THE DEPHOSPHORYLATED FORM WILL
INHIBIT MILD ACTIVITY OF THE
MOUSE TRANSPORTER.
AND WE APPROACH THIS BY
DETERMINING THE STRUCTURE OF THE
TRANSPORTER TOGETHER WITH E2A.AND THIS IS WHAT IT LOOKS LIKE.
SO THIS IS THE COMPLEX.
AND MOUSE'S TRANSPORTER IS IN
THE INWARD FACING STATE AND WE
SEE TWO E2A MOLECULES WHICH ARE
SHOWN IN PURPLE HERE BIND TO THE
MILD K SUBUNITS.
AND BEFORE OUR STRUCTURE,
ACTUALLY THERE WERE MANY -- HAS
ALREADY IDENTIFIED MUTATIONS IN
THE MODEL TRANSPORTER WILL
ESCAPE E2A INTERACTION.
AND WE LOOK WHERE THE
LOCATIONS THOSE MUTATIONS WHICHARE SHOWN I N GREEN AND YELLOW
HERE WERE ALL LOCATED AS
INTERFACE WITH E2A.
SO THIS GIVES US CONFIDENCE
ABOUT THE ACCURACY OF THISCOMPLEX.
ALSO, THE SITE OF
PHOSPHORYLATION IS AGAIN RIGHT
AT THE INTERFACE.
IT ACTUALLY FORMS A HYDROGEN
BOND WITH THE MILD K SUBUNIT.
SO THIS GIVES, THIS EXPLAINS WHY
PHOSPHORYLATION OF THIS
HISTIDINE WILL BECOME, WILL
DEASSOCIATE, WILL MAKE THIS
COMPLEX UNSTABLE SO THIS
INTERFACE IS NOT COMPATIBLE WITH
PHOSPHORYLATED E2A.
SO ONLY IN THE DEPHOSPHORYLATION
FORM E2A WILL BIND THE
TRANSPORTER AND INHIBIT ITS-FUNCTION.
SO HOW DOES E2A INHIBIT MOUSE
TRANSPORTER.WE REVIEW WHAT THE TRANSPORTER
HAS TO DO TO ENABLE -- BASIC THE
RIDGED BODY ROTATION INVOLVES
THE TRANSMEMBRANE DOMAIN AND THE
MBD.
NOW WE HAVE TWO E2A MONITORS
COME AND BIND TO THE MILD K
SUBUNITS.
AND EVERY E2A SIMULTANEOUSLY
INTERACTING WITH THE REGULATORY
DOMAIN OF ONE SUBUNIT AND THE
MBD DOMAIN OF OTHER SUBUNITS.
THEY BASICALLY LOCK THE
TRANSPORTER IN THIS INWARD
FACING STATE.
NOW THE MBD CAN NO LONGER ROTATE
IN TO FORM THE OUTWARD FACING
STATE.
SO BASICALLY -- INHIBITOR IT
PREVENTS MILD UPTAKE AND ATP
HYDROLYSIS BY PREVENTING THE
CONFIRMATION CHANGE.THIS TRANSPORT HAS TO GO THROUGH
TO DO THESE THINGS.
SO ANOTHER INTERESTING THING
ABOUT E2A IS ACTUALLY BY
OBSERVATIONS NATURALLY IT HAS
THIS TWO FORMS.
THE FULL LENGTH E2A.
THE OTHER LINE IS TRUNCATED
E2A -- WHEN PEOPLE STUDY HOW E2A
FUNCTION WE NOTICE THE DIVERSION
OF E2A HAS NO PROBLEM WITH
INTERACTING WITH THE SOLUBLE
PARTNER.
HOWEVER THIS DEFICIENT IN
INTERACTING WITH TRANSMEMBRANE
SUBUNITS.SO IN OUR CRYSTAL STRUCTURE, WE
HAVE E6789 2A BUT ONLY SEE
RESIDUES FROM 19 -- SO WHEN YOU
LOOK AT THE POSITION OF THE
FIRST RESIDUE WE SEE THEY ARE
POINTING RIGHT TOWARDS THE
MEMBRANE.
AND ACTUALLY WAS -- MANY YEARS
AGO HAS PROPOSED THIS IDEA.
MAYBE THIS TERMINAL REGION
ALTHOUGH MOST PEOPLE DON'T SEE
IN STRUCTURE HAS AN IMPORTANT
FUNCTION.THEY MAY FUNCTION AS A MEMBRANE
ANCHOR TO BRING E2A TO THE
MEMBRANE SURFACE TO ENABLE TO AN
HANCE THE INTERACTION WITH
TRANSPORTERS.MORE STRUCTURES DETERMINE OF THE
PEPTIDE AT THE END TERMINAL
REGION SHOWS IT FORMS A HELIX
WITH ALL THE HYDROPHOBIC LINING
ON ONE SURFACE OF THE HELIX.
SO ALSO SUGGESTS THIS MIGHT BE
THE FUNCTIONS OF THE END
TERMINAL REGION.
SO WE HAVE THIS HYPOTHESES BY A
VERY SINGLE EXPERIMENT.
SO WHAT WOULD BE WE RECONSTITUTE
OUR TRANSPORTER INTO WHAT WE
CALL THE NANO DISK LIKE A
MEMBRANE DISK WITH THE
TRANSPORTER IMBEDDED TO THAT.
THEN WE CAN ADD MDP AND THE
MOUSE FROM ONE SIDE AND -- ON
THE OTHER SIDE THEN WE MONITOR
THE ABILITY OF THE E2A INHIBIT
EPA HYDROLYSIS.
WE CAN ACHIEVE ABOUT 90%
INHIBITION COEFFICIENT ABOUT
1.5.
SO THIS IS A CONSISTENT WITH WE
NEED TWO E2A'S TO BIND TO THE
TRANSPORTER.-WITH THE TRUNCATED VERSION OF
E2A WE ALSO CAN INCLUDE IT BUT
IT TAKES A LOT MORE PROTEIN.
SO NOW THE -- IS ALMOST 100
MICRO MOLARS IN COMPARISON TO
1.6.
THE ONLY DIFFERENCE BETWEEN THE
TWO EXPERIMENTS IS THE TERMINAL
REGIONS.THIS SUPPORTS THE IDEA THAT THE
TERMINAL REGION OF THE E2A
FUNCTIONS THE MEMBRANE ANCHOR.
SO WITH THAT I'M GOING TO
SUMMARIZE WHAT WE HAVE LEARNED
ABOUT THIS SYSTEM.
SO THE TRANSPORTER FUNCTION
THROUGH ALTERNATING ACCESSMECHANISM.
SO IN THE A SUBJECT OF ANY
SUBSTRATE THE TRANSPORTERRESTING IN THE IN WARD FACING
CONFIRMATION WITH TWO MBD
SEPARATE FROM EACH OTHER.
THEY WILL NOT AWAIT ATP.
THE PRESENCE OF THE BINDING
SUBSTRATE WILL STABILIZE THE
BINDING PROTEIN IN THE CLOSE
CONFIRMATION AND THE CLOSE
CONFIRMATION BINDING PROTEIN
WILL INTERACT WITH THE
TRANSPORTER BRING THE TWO MBDS
CLOSE TO EACH OTHER, CLOSE
ENOUGH.
NOW ATP BINDING WILL CONVERT
THE -- TO OUTWARD FACING STATE
WHERE MALTOSE IS DELIVERED INTO
THE MEMBRANE AND READY TO
HYDROLYZE.ATP HIGH DRUG SIST -- TO THE
INWARD FACING STATE TO THE SAME
TIME RELEASING THE MOUSE INTO
THE CELL.
WHEN THE PREFERRED SUGAR
SUFFICIENT AS GLUCOSE IS PRESENT
IN THE MEDIA, IT WILL BE
TRANSPORTED THROUGH THE PTS
SYSTEM -- OF THE GLUCOSE WILL
BECOME DEPHOSPHORYLATED AND
DEPHOSPHORYLATED E2A WILL
INTERACT WITH THE TRANSPORTER
STABILIZE IT IN THE INWARD
FACING STATE.
NOW BINDING PROTEIN AND ATP CAN
NO LONGER -- THE TRANSPORTER
CYCLE.I SHOULD ALSO POINT OUT IN E.
COLI ONLY THE GLUCOSE SPECIFIC
E2A WILL VIEW THE REGULATION FOR
THE CARBON CATABOLIC REPRESSION.
HOWEVER, ANY PTS SUGARS THERE
ARE PROBABLY ABOUT 20 OF THEM.
ANY UPTAKE OF THE PTA SUGAR WILL
CONSUME THE PHOSPHATE FROM THE
SAME P -- SO BY DOING THAT YOU
CHANGE THE BALANCE BETWEEN THE
PHOSPHORYLATE AND
DEPHOSPHORYLATE FORM OF THIS
GLUCOSE SPECIFIC E2A.
SO THE PRESENCE OF ANY -- SUGAR
WILL ENABLE INDUCE
[INDISCERNIBLE]-SO WITH THAT I NEED TO THA NK
PEOPLE WHO DID THE WORK.
MIKE -- A PERMANENT SCIENTIST IN
MY LAB.
HE DETERMINED THE TWO CRYSTAL
STRUCTURES.
THOSE ARE THE OUTWARD FACING
STATE AND THE PRETRANSLOCATION
STATE -- IS A GRADUATE STUDENT
IN MY LAB WHO JUST COMPLETED THE
STRUCTURE OF THE E2A INHIBITOR
TRANSPORTER -- IS MY FIRST POST
DOC WHO DETERMINED THE INWARD
FACING STRUCTURE.AND THIS HAS BEEN A LONG TERM
COLLABORATION WITH -- CONTINUOUS
FUNDING FROM NIH WAS EXTREMELY
IMPORTANT FOR THIS PROJECT AS
WELL AS LATER FUNDING FROM --
HHMI.-THANK YOU.-[APPLAUSE]
>> I'M SURE SHE WILL BE HAPPY TO
ANSWER SOME OF YOUR QUESTIONS.
>> REALLY BEAUTIFUL WORK.->> THANK YOU.
>> THE QUESTION IS REALLY
SPECIFICITY OF THE PERI
PLASMIC -- SYSTEM.
NOW THE MD -- HOWEVER THERE ARE
A LOT OF BIEBLEDDING PROTEINS IN
THE PARAPLASMIC.
SO HOW IS SPECIFICITY ENFORCED.
PREDICTION IS THE ATP BINDING
ACTUALLY STRENGTHENS, HAS A ROLE
NOT JUST HOPING OR CLOSING
INWARD OUTWARD PLAYING A ROLE IN
SPECIFICITY.>> SPECIFICITY YOU MEAN FOR TH E
TRANSPORTER SUBSTRATE.->> EXACTLY.
>> YES.
SO FOR THE MALTOSE SYSTEM WE
ACTUALLY KNOW PRECISELY I DIDN'T
HAVE TIME TO TALK ABOUT IT.
WHAT'S KNOWN IS THE SPECIFICITY
OF MALTOSE TRANSPORTER IS BY THE
BINDING PROTEIN.
SO THIS TRANSPORT -- LOOK THE
GLUCOSE FROM TWO GLUCOSE TO-SEVEN GLUCOSE -- SO IT'S VERY
SPECIFIC.
SO MALTOSE BINDING PROTEINS
SELECTIVELY BINDS THOSE SUGARS.
WE ALSO KNOW SOME CHOORGZ --
CANNOT BE TRANSPORTED.
SO MY WORK I DIDN'T HAVE TIME TO
TALK ABOUT.WE NOW WHAT HAPPENS IS THE SUGA R
HAS A POLARITY IT HAS A REDUCED
END AND A NON-REDUCING END.MALTOSE BINDING PROTEIN WILL
BIND THE SUGAR AT THE EITHER
REDUCING END BIND THE SUGAR FROM
THE -- BUT BASICALLY THE
SUGAR -- FROM THE BINDING
PROTEIN INTO THE MEMBRANE AND
BOTH SIDES ARE RESTRICTED.
SO THE SUBSTRATE SPECIFICITY FOR
THE MALTOSE SYSTEM IS VERY --
THE BINDING PROTEIN AND THE
TRANSMEMBRANE SUBUNIT.>> THAT'S VERY NICE.
HOWEVER DOES ATP PLAY A ROLE IN
SELECTIVITY.>> ATP LAYS NO ROLE, THAT'S
CLEAR.-IT'S LIKE THE -- DRIVING THE
SYSTEM.>> IF THAT'S THE CASE THE
QUESTION'S ALSO THAT THE
INTERFACE BETWEEN -- THE
TRANSMEMBRANE PORTION.
FROM YOUR DIAGRAM THE INTERFACE
CHANGE AT DIFFERENT STATES.
BUT IT'S NOT JUST A --
MECHANICAL MOTION IT'S MORE --
>> YES.
SO SHE ASKED ABOUT WHAT ABOUT
INTERFACE BETWEEN THE
TRANSMEMBRANE SUBUNITS AND THE
MBD.
SO WHAT WE SEE IS THE MBD HAVE A
PRETTY HYDROPHOBIC CLEAVE ON THE
SURFACE AND -- HAS A HELIX
COMING OUT.INSIDE THIS.
WE CALL THIS -- SO DOING
DIFFERENT CONFIRMATIONAL CHANGES-ESPECIALL Y WITH THIS BALL FROM
THE TRANSMEMBRANE DOMAIN WILL
ROTATE INSIDE CLIFF.-ONE SIDE OF THE CLIFF IS QUITE
FLEXIBLE.YOU CAN ADJUST ITS SIZE.
SO BY DOING IT IT WILL ENABLE
DIFFERENT CONFIRMATIONAL CHANGES
BUT IT MAINTAINS A CLOSE
CONTACT.
AND -- AT PRESENT POSITIONS IS
ALSO CRITICAL SO THERE'S A
CERTAIN -- AND THEN CERTAIN
FLEXIBILITIES BY DOING THAT IT'S
ABLE TO MAINTAIN HIGH AFFINITIES
BUT ALSO ALLOW RELATIVE MOTION
IN DIFFERENT CONFIRMATIONS.
>> THANK YOU.
>> THANK YOU, AN INTERESTING
TALK -- THROUGH THE WHOLE
TRANSPORT CYCLE.I HAVE A QUESTION REGARDIN G THE
INWARD CONFIRMATION AND OUTWARD
CONFIRMATION.IF I UNDERSTAND YOU CORRECTLY ,
IF YOU HAVE ATP HYDROLYZED ATP
BINDING POCKET, THAT DOESN'T --
CONFIRMATION CHANGE WHICH WILL
BE INWARD FROM OUTWARD --
OUTWARD TO INWARD.
>> THAT'S CORRECT.->> SO YOU NEED TO HAVE BOTH THE
SUBSTRATE AND THE ATP AT THE
SAME TIME.->> PRECISELY.>> WHAT WOULD BE T HE NEXT STEP
AFTER THIS?
HOW WILL THE SEQUENCE OF EVEN
THE ATP -- AFFINITY OF THE
TRANSPORTER.-I'M ASKING THIS QUESTION IN
REGARDS TO THE EXPORT WHERE YOU
HAVE -- BUT WHAT WILL BRING A
CHANGE IN THE AFFINITY TO THE
BINDING SITE.>> I DON'T QUITE UNDERSTAND T HE
QUESTION.YOU MEAN WHAT WILL HAPPEN AFTER
ATP IS BOUND IN THE --
>> YES.
>> SO WE KNOW ATP WILL BE
HYDROLYZED.
WE ALREADY SEE THE FORMATION OF
THE TRANSITION STATE OF
HYDROLYSIS.%THEN SO THE BOND BETWEEN ATP A ND
THE PHOSPHATE WILL BE BROKEN.
THEN WHAT HAPPENS IS PRETTY MUCH
WE KNOW FOR SURE ORGANIC
PHOSPHATE WILL BE RELEASED.
THEN, THEN WHETHER IT'S THE
RELEASE OF THE INORGANIC
PHOSPHATE ALONE OR THE RELEASE
OF THE INORGANIC PHOSPHATE PLUS
ADP TO OPEN UP THE DIMER WE
STILL DON'T KNOW THAT DETAIL.
>> TWO DIFFERENT CONFIRMATIONS
YOU BASICALLY NEED BOTH ATP AND
THE SUBSTRATE OR YOU DON'T HAVE
ANY OF THEM AND YOU CHANGE --
FROM OUTWARD TO INWARD
BASICALLY.>> THERE ARE TWO BASIC
CONFIRMATIONS.-INWARD AND OUTWARD.
THERE'S AN ENERGY BARRIER IN
BETWEEN.
IN THE FORWARD CYCLE THE ENERGY
BARRIER IS OVERCOME BY THE
BINDING ENERGY FROM BOTH THE
SUBSTRATES AND ATP.
SO YOU NEED THE SUBSTRATES TO
BIND AND ATP BIND NOW YOU CAN GO
TO THE OUTWARD FACING STATE.
ONCE WE REACH OUTWARD FACING
STATE IT HAS NO CHOICE BUT TO
HYDROLYZE ATP BECAUSE -- THEN
THE HIGH HYDROLYSIS -- SO THE
INWARD FACING STATE.
THIS REVERSE CYCLE BUT NOW BY
REVERSING THE CYCLE WE NOW
EXPOSE THE SUBSTRATE INTO THE
CYTOPLASM NOW AND THE BINDING
SITE HAS VERY LOW AFFINITY FOR
THE SUBSTRATE.THIS IS SECTED TO BE INTO TH E
CELL AND WILL BE DIFFUSED OUT
AND DEMETABOLIZED.->> THANK YOU.
>> BEFORE I ASK THE LAST
QUESTION I WANT AN ANNOUNCEMENT.
THERE'S A RECEPTION AFTER THE
Q&A.-PLEASE COME AND JOIN US.
SO MY QUESTION IS YOU MENTIONED
THE LIQUID [INDISCERNIBLE]
ARE THEY COMING FROM THAT.
>> YES.
SO FUNCTIONALLIZE MODEL TRANSFERDOESN'T SE EM TO CARE WHERE THE
LIPIDS, THEY ALL WANT LIPID
PRESENCE.-IT'S REALLY WELL -- BUT THE
STRUCTURALLY REDUCE THE ONE --
FORWARD BOUND TO A CERTAIN
POSITION ON THE PERIPHERAL OFTHE TRANSPORT ER.
YOU PHASE INTO A SURFACE
DEPRESSION BETWEEN TWO
TRANSMEMBRANE HELIXES AND BECAME
PART OF THE STRUCTURE.
BUT IN DIFFERENT CONFER MAKES WE
ALL SEE THE SAME LIPIDS.
SO YOU CAN IMAGINE THIS BECOMES
PART OF THE STRUCTURE SO MAYBE
STABILIZE THE TRANSPORT BEING
THE CULTURE -->> THANK YOU AGAIN