>> GOOD MORNING, EVERYONE, AND
WELCOME TO ANOTHER GREAT EDITION
OF THE BIOWULF SEMINAR SERIES.
IN FACT, THIS YEAR WE ARE
CELEBRATING THE 25TH ANNIVERSARY
OF BIOWULF.
[APPLAUSE]
AND WE HAVE A GREAT LINEUP OF
SCIENTIFIC TALKS FOR THIS YEAR'S
SEMINAR.
WE ALSO HAVE SPECIAL VISITORS
ATTENDING OUR TALK TODAY.
AND TODAY SPECIFICALLY, WE ARE
HONORED TO HAVE DR. JULIANA
MARTINEZ FIESCO AS OUR SPEAKER,
SHE RECEIVED HER Ph.D. FROM
FLORIDA STATE UNIVERSITY WHERE
SHE STUDIED GLOBAL KINASE
MECHANISM OF ACTION AND
ACTIVATION AND ALSO TRAINED AS A
BIOCHEMIST.
IN 2017 SHE JOINED
DR. PING JEN'S LAB IN THE KINASE
SECTION -- CENTRAL KINASE
SECTION IN NATIONAL CANCER
INSTITUTE?
FREDERICK.
THIS WAS FOR HER POSTDOCTORAL
TRAINING, WHERE SHE ALSO STUDIED
A SET OF KINASES AND TRAINED AS
A STRUCTURAL BIOLOGIST.
SHE RECENTLY TRANSITIONED TO A
STAFF SCIENTIST POSITION WITH
PI -- PINGJEN'S LAB, HER
SPECIFIC INTEREST IS TO
UNDERSTAND THE STRUCTURE
FUNCTION RELATIONSHIP IN ENZYMES
TO UNDERSTAND THEIR MECHANISM OF
ACTION, TO GAIN SIGHT INTO
DIFFERENT DISEASES.
SHE IS AN AVE. INDIVIDUAL --
USER AND HER TALK TODAY IS
TITLED STRUCTURAL INSIGHTS INTO
THE MONOMER ARE DIMER TRANSITION
MEDIATING BY RAS BINDING.
[APPLAUSE]
HAD.
>> GOOD MORNING, EVERYONE, THANK
YOU FOR THE OPPORTUNITY SHARE
OUR WORK TODAY.
AS HE MENTIONED, TODAY I'M GOING
TO SHARE SOME STRUCTURAL WORK WE
HAVE DONE IN THE LAB IN
SPECIFICALLY IN BRAF PROTEINS
AND WE SPECIFICALLY CHOSE THIS
PROJECT BECAUSE WE REALLY RELY
ON BIOWULF RESOURCES TO BE ABLE
TO DO OUR ANALYSIS ESPECIALLY
THE STRUCTURAL WORK PART.
SO I JUST WANT TO MAKE SURE THIS
IS WORKING OKAY.
AS YOU MAY KNOW, THE WILL RAS
PATHWAY PLAYS A VERY IMPORTANT
ROLE IN THE TRANSMISSION OF
GROWTH SIGNALS BUT ACCOUNTS ALSO
A CONTRIBUTOR CONTRIBUTOR OF
HUMAN DISEASE STATES, HAVING
SPECIFICALLY MUTATIONS IN RAS
PROTEINS AND BRAF -- AND RAS
PROTEINS BEING THE MAJOR DRIVERS
OF MANY DIFFERENT TYPES OF
CANCERS.
BECAUSE OF THIS, THESE PROTEINS
ARE THE TARGET SPORE THERAPEUTIC
INTERVENTION, BUT IN ORDER TO DO
THAT, IT'S VERY IMPORTANT TO
UNDERSTAND THE MECHANISTIC
DETAILS OF HOW THE SIGNALS ARE
TRANSMITTED THROUGH THE PATHWAY.
SO OUR LAB HAS BEEN FOCUSED ON
UNDERSTANDING THE STRUCTURE OF
ONE OF THESE PROTEINS IN ORDER
TO UNDERSTAND HOW THEY AK -- HOW
THE ACTIVATION OF THESE PROTEINS
HAPPEN AND HOW THE SIGNALS ARE
TRANSMITTED THROUGH THE PATHWAY.
SO WE ARE MAINLY FOCUSED ON THE
STRUCTURAL BIOLOGY PART OF THE
PATHWAY, SO WE WANT TO DETERMINE
THE DIFFERENT STRUCTURES OF BRAF
PROTEINS IN DIFFERENT COMPLEXES.
SO BRAF PROTEINS, THERE ARE
THREE MEMBERS IN THE BRAF
FAMILY.
WE HAVE ARAF PROTEIN, BRAF AND
CRAF PROTEINS, BUT BRAF AND
CRA -- AND CRAF PROTEINS THE
MAIN DRIVERS MUCH HUMAN DISEASES
SPECIFICALLY CANCER.
ALL THE MEMBERS OF THE FAMILY
HAVE A SIMILAR DOMAIN
ORGANIZATION, SO YOU CAN THINK
OF IT AS IF IT HAS AN END
TERMINAL REGULATORY DOMAIN AND
FROM THIS HAS A DOMAIN THAT IS
CALLED THE RAS BINDING DOMAIN
THAT WE'RE GOING TO CALL IN THIS
PRESENTATION RBD AND CYSTEINE
RICH DOMAIN, CRD.
THIS IS THE REGULATORY PART OF
THE PROTEIN.
AT THE C TERMINALS WE HAVE THE
KINASE DOMAIN AND THIS IS THE
PART OF THE PROTEIN THAT
ACTUALLY CARRIES OUT THE
CATALYTIC FUNCTION SO THE -- SO
THE NEXT PROTEIN IN THE CHAIN
WHICH IS MEK.
THESE PROTEINS ARE FOUND IN AN
INHIBITORY, OUTER INHIBITORY
STATE AND IT'S BASICALLY
INTERACTIONS BETWEEN THE N
TERMINAL REGULATORY DOMAINS AND
THE C TERMINAL DOMAIN THAT
MAINTAIN THE STATE BUT YOU CAN
ALSO SEE THAT INTERACTION WITH
14-3-2 PROTEINS THAT FORM A
DIMER AND HELP STABILIZE THE
OUTER STATE.
ONCE THE PROTEIN RECEIVES
SIGNALS TO ACTIVATE THE PATHWAY,
WHAT HAS HAPPENED IS THAT THE
PROTEIN, THE BRAF PROTEIN IN
THIS CASE INTERACTS WITH PROTEIN
THROUGH THE BINDING DOMAIN OF
THE RBD DOMAIN.
WHEN THIS INTERACTION HAPPENS,
WE ARE GOING TO HAVE SOME
CONFORMATIONAL CHANGES IN THE
PROTEIN THAT ARE GOING TO
PRODUCE DIMER ZAIFTION THE
PROTEIN -- DIMERIZATION OF THE
PROTEIN AND IN TURN PRODUCE
ACTIVATION OF THE PROTEIN.
SO IT HAS BEEN KNOWN FOR
DIFFERENT STUDIES, MAINLY ARE
CRYSTAL STRUCTURES OF JUST
CONTAINING THE KINASE DOMAIN OF
THE R PROTEINS, NEED TO DIMER
EYES IN ORDER TO BECOME ACTIVE.
THERE ARE SOME STRUCTURAL
ARRANGEMENTS THAT NEED TO HAPPEN
WHEN RAS BINDING TO RAF IN ORDER
FOR THE DIMERIZATION TO TAKE
PLACE AND THEN THE PATHWAY CAN
BE ACTIVATED.
SO WHAT WE REALLIMENT TO
UNDERSTAND IS HOW THE BINDING OF
RAS TO RAF IS GOING TO PRODUCE
THE TRANSITION THAT IS REQUIRED
TO GO FROM MONOMER TO DIMER.
SO WE WANTED TO INVESTIGATE THIS
RAILROAD STRUCTURALLY.
BEFORE I CONTINUE EXPLAINING
WHAT WE DID, EU79 TO MENTION
THAT THIS WORK HAS BEEN DONE IN
COLLABORATION WITH DR. MAURICE
SONT AT THE LABORATORY OF
DEVELOPMENTAL SIGNALLING AND
FOLLOWED DEBBIE DUR RANT, SO
THEY ARE VERY EXPERTS LIKE IN
ALL THE RAS AND RAF BIOLOGY AND
OUR LAB HAS MORE EXPERTISE IN
THE -- AND OUR LAB HAS MORE
EXPERTISE IN THE STRUCTURAL
BIOLOGY PART, SO IT WAS A VERY
COMPLEMENTARY WORK BETWEEN THE
TWO LABS.
WE MAINLY USE A ELECTRIC CALLED
CRYOMICROSCOPY TO SIGN THE
STRUCTURES OF BRAF PROTEIN IN
THESE DIFFERENT COMPLEXES AND WE
WERE ABLE TO DETERMINE THREE
DIFFERENT STRUCTURES.
TWO STRUCTURES THAT CORRESPONDS
TO RAF IN ARE THE STATE, ONE HAS
MEK, ONE DOESN'T HAVE MEK AND
DIMER STRUCTURE OF THE PROTEIN.
BRING MOVE FORWARD TO SHOW YOU
THE STRUCTURE, I JUST WANTED TO
GIVE A BRIEF DESCRIPTION OF WHAT
THE CRYO-EM PROCESS LOOKS LIKE.
SO NORMALLY YOU YOU'RE PHI YOUR
PROTEIN, YOU WANT TO HAVE YOUR
PROTEIN TO BE HIGHLY PURE AND BE
VERY HOMOGENEOUS, THEN YOU APPLY
THIS PROTEIN IN WHAT IS CALLED A
CRYO-EM GRID.
SO THE CRYO-EM GRID IS JUST
WHERE YOUR PROTEIN IS GOING TO
BE DEPOSITED SO BUT NORMALLY
CARBON, YOU'LL SEE THIS CARBON
LAYER HAS SOME TINY HOLES.
SO THE IDEA IS THAT WHEN YOU
APPLY THE SAMPLE TO THIS GRID,
YOU CAN BLOT THE EXCESS SOLUTION
AND THEN YOU FREEZE IT VERY
FAST.
WHAT YOU END UP HAVING IS THAT
THE IN HOLES, IN THE CRYO-EM
GRID, YOU FORM A VERY FINE LAYER
OF ICE, AND IN THIS LAYER OF
ICE, YOU HAVE DIFFERENT
PARTICLES OF YOUR PROTEIN, SO
YOU MAYBE CAN APPRECIATE IT
BETTER IN THIS DIAGRAM HERE.
SO YOU'RE GOING TO HAVE YOUR
PROTEIN LIKE IN THIS VERY TINY
LAYER OF ICE, AND THE PROTEIN IS
GOING TO BE LIKE SITTING IN
DIFFERENT ORIENTATIONS.
SO WHAT YOU DO IS THAT YOU IMAGE
THESE PARTICLES IN THIS ICE WITH
AN ELECTRON BEAM AND THEN YOU
DETECT, OBVIOUSLY WITH ELECTRON
DETECTOR, AND WHAT YOU
UNDERSTAND UP COLLECT SG A
SERIES OF IMAGES THAT CONTAIN 2D
PROJECTIONS OF YOUR PARTICLE, IN
THIS CASE YOUR PROTEIN.
AND WHAT YOU DO IS THAT YOU
EX-TRABLGHT THESE 2D CLASSES OR
2D I AM EXPHADGES TRY TO USE
THIS 2D IMAGE TO BACK PROJECT
AND RECONSTRUCT THE
THREE-DIMENSIONAL STRUCTURE OF
THE PROTEIN.
TH T THIS IS WHERE YOU NEED A
LOT OF COMPUTATIONAL RESOURCES
TO DO THAT AND THIS IS WHERE
BIOWULF WAS VERY IMPORTANT FOR
US IN THIS PROJECT.
SO PROBABLY MOST OF THE PEOPLE
HERE KNOW THAT BIOWULF HAS LIKE
A LOT OF RESOURCES THAT ARE
DEDICATED TO THE CRYO-EM DATA
ANALYSIS.
I THINK I COUNT PROBABLY 20
DIFFERENT SCIENTIFIC PROGRAMS
THAT YOU HAVE IN BIOWULF, AND
FOR THIS PROJECT, WE MAINLY USE
RE -- RELION.
WE DID THREE DIFFERENT PURE
FIXES.
THERE ARE TWO PURE FIXES THAT WE
USE -- PURIFICATIONS THAT WE
USE.
ALL THE PURIFICATIONS WERE DONE
FROM MAMMALIAN CELLS THAT WERE
ALREADY EXPRESSING A FORM OF THE
BRAF PROTEIN THAT HAD HALO T
TAG.
THE PROTEINS YOU'LL SEE IN THE
COMPLEX WERE ENDOGENOUS PROTEINS
IN THESE MAMMALIAN CELLS.
SO WE HAVE TWO PURIFICATIONS
THAT WERE DONE IN SERUM DEPLETED
CELLS AND WE ISOLATED FROM THESE
TWO SAMPLES PROTEIN COME PLEKS
THAT CONTAIN JUST ONE MONOMER OF
BRAF AND THEN WHEN WE TREATED
THE SAMPLE -- OR THE CELLS,
SORRY, WITH BRAF INHIBITOR THAT
PATROLMAN THE DIMER FORMATION,
WE WERE ABLE TO ISOLATE A -- --
SO YOU CAN SEE HERE, ON THE
RIGHT IS THE SDS GELS THAT
ALLOWED US TO IDENTIFIES WHAT
PROTEINS WERE PRESENT AND ALSO
THE PURITY OF THE COMPLEX THAT
WERE PURIFIED.
SO YOU CAN SEE HERE, IN THE
FIRST TWO GELS THAT I'M SHOWING
HERE, WHERE WE ISOLATED THE
MONOMERIC OF WE HAVE THE
PRESENCE OF BRAF AND 14-3-3 AND
ALL THE PRESENCE OF MEK.
IN THE OTHER ONE WHEN WE TREATED
THE CELLS WITH THE KINASE
INHIBITOR SPECIFICALLY BRAF
INHIBITOR, WE MAINLY ONLY HAVE
BRAF AND 14-3-3.
SO WE'LL TAKE A LOOK FIRST AT
THE SAMPLE WHERE WE PURIFY THE
DIMERIC FORM OF BRAF, WE CAN
SEE, I'M SHOWING YOU HERE
REPRESENTATIVE TO THE CLASSES OR
TO THE IMAGES THAT WE USED TO
RECONSTRUCT, TO FIND A
RESOLUTION OF THE STRUCTURE OF
THE BRAF.
SO YOU CAN SEE HERE THAT WE HAVE
THIS BIG BLOCK HERE WHERE WE CAN
FIT THE KINASE DOMAIN, AND HERE
AT THE BOTTOM, WHERE WE CAN FIT
14-3-3 PROTEINS.
SO THIS IS WHAT IS SHOWN YOU
HERE IN THESE REPRESENTATIONS.
SO THE FIRST THING I WANT TO
MENTION IS THAT IN THE DIMER
FORM, THE N TERMINAL REGION, THE
REGULATORY REGION THAT CONTAIN
THE R DOMAIN AND CDD DOMAIN WERE
NOT VISIBLE, SO THE PRESENCE OF
THE MEMBRANE WAS REQUIRED TO
STABILIZE THAT IN THE DIMER
OTHERWISE IT SEEMS TO BE VERY
FLEXIBLE AND WE WERE ABLE TO
RECONSTRUCTED IT.
LY.
WE CAN SEE HERE THAT WE FORM IN
THIS COMPLEX THE CHARACTERISTIC
BACK TO BACK DIMER, I SHOWED
BEFORE WAS DETERMINED BY X-RAY,
AND WE CAN SHOW THE PRESENCE IN
THE DIMER AND WE CAN ALSO SEE
THAT THIS -- STRUCTURE IS
PRESENT THIS AN ACTIVE
CONFIRMATION.
WE CAN MAINLY SEE THAT BECAUSE
OF THE POSITION OF THESE
HELICESR THESE HEL I CES CAN
ADOPT VERY SPECIFIC FORMATION
WHEN THE PROTEIN IS ACTIVE AND
WHEN THE PROTEIN IS INACTIVE.
IN THIS DIMER STRUCTURE, WE
DEFINITELY SEE IT IN
CONFIRMATION -- IN THE
CONFORMATION INDICATING THAT THE
PROTEIN IS ACTIVE.
WE CAN SEE EACH -- TREN GAUGE
EACH COPY OF THE 14-3-3-3 WHICH
IS A DIMER, EACH INTERACTS WITH
14-3-3 WITH THE PHOSPHORYLATION
SIDE WHICH IS -- WHICH IS PS729.
WE WANT TO SEE HOW WE CAN GET TO
THIS DIMER CONFORMATION GOING
FROM THE MONOMER CONFORMATION.
ONE THING I FORGOT TO MENTION IS
THAT IT INVOLVES ACTIVE SITES OF
THE KINASE DOMAIN, WE SEE THE
PRESENCE OF THE KINASE INHIBITOR
THAT WE USE TO FORM THE COMPLEX,
AND THIS IS AN ATP ANALOG
BINDING TO THE ACTIVE SIDE OF
THE PROTEIN, SO THIS INDICATES
THAT PROTOMERS OF THE BRAF CAN
PERFORM CAT LIS AT THE SAME TIME
SO BOTH ACTIVE CONFORMATIONS OF
THE PROTEIN.
SO IF WE LOOK AT THE MONOMER
STRUCTURES, HERE AGAIN I'M
SHOWING YOU THE 2D CLASSES THAT
WERE USED TO RECONSTRUCT THE
THREE-DIMENSIONAL STRUCTURE OF
THE PROTEIN R TWO COMPLEXES, ONE
HAS THE PRESENCE OF MEK WHICH
WAS RESOLVED AT 3.27 -- AT 3.7
RESOLUTION AND OTHER ONE ABSENCE
OF MEK, WHICH IS 4.1.
YOU CAN SEE ABSENCE OF MEK IS A
LITTLE LOWER SO IT MAY INDICATE
THAT THE PRESENCE OF MEK HELPS
TO STABILIZE THE CONFORMATION OF
THE COMPLEX.
SO IF WE SEE THE ATOMIC
STRUCTURE, WE CAN SEE THAT WE
HAVE AT THE CENTER OF THE --
FIRST OF ALL, BOTH STRUCTURES
ARE ALMOST THE SAME IN THE
PRESENCE OR ABSENCE OF MEK.
THE KINASE DOMAIN AND RBD AND
CRD ARE IN THE SAME CONFORMATION
ORIENTATIONS, IT SEEMS THAT THE
MEK HELPS TO STABILIZE THE
COMPLEX.
OTHERWISE, THEY ARE LIKE
IDENTICAL.
WE CAN SEE THAT THE CRD DOMAIN
IS AT THE CENTER OF THE
INHIBITORY STATE, SHOWN HERE IN
VIOLET, AND WHAT YOU CAN SEE
HERE IS THAT IT MAKES
INTERACTIONS WITH THE 14-3-3
DIMER AND WITH THE KINASE
DOMAIN.
SO IT SEEMS THAT IT REALLY HELPS
TO MAINTAIN, AT THE CENTER OF
THE OUTER STATE OF THE PROTEIN.
WHEN MEK IS PRESENT WE CAN SEE
THAT IT INTERACTS WON'T KINASE
DOMAIN OF THE BRAF, SO IT'S LIKE
KINASE-KINASE INTERACTION BUT
MEK DOESN'T INTERACT WITH THE
REGULATORY PART OF THE BRAF
PROTEIN.
IF WE LOOK AT THE KINASE DOMAIN
OF THE PROTEIN, WHICH IS SHOWN
HERE IN BLUE F WE JUST FOCUS TO
THAT PART, THE FIRST THING THAT
WE CAN OBSERVE IS THAT IN OUR
STRUCTURES, THE ATP BINDING
POCKET IS EMPTY, SO THERE IS NO
ATP BOUND IN OUR STRUCTURES, AND
WHERE WE CAN SEE THAT THIS
KINASE DOMAIN ADOPTS THE
INACTIVE CONFORMATION.
SO AGAIN, WE CAN SEE THAT
BECAUSE THE POSITION OF THESE
ALPHA C HELIX SHOWING IN RED IS
IN WHAT IS CALLED THE OUT
POSITION, SO IT IS ACTIVE
CONFORMATION AND MAINLY IT IS
BECAUSE WHEN THESE ALPHA HELIX
IS IN THE OUT CONFORMATION WHAT
IS CALLED REGULATORY SPINE IS
BROKEN BECAUSE THIS IS
MISALIGNED.
THE OTHER THING AWE OBSERVED IS
THAT EVEN THOUGH OUR STRUCTURE
WAS DONE IN THE ABSENCE -- OR
WAS IN THE ABSENCE OF MEK,
ALMOST THE SAME CONFORMATION
THAT WHEN ATP ANALOGS ARE BOUND
TO THE PROTEIN.
AND THIS WAS IMPORTANT BECAUSE
IN THIS STRUCTURE, WE CAN SEE
THAT WHAT WE CALL THE N LOBE AND
C LOBE END UP LIKE A MORE
COMPACT CONFORMATION AND IN THIS
CONFORMATION, IT'S IMPORTANT TO
FORM A DIMER ARE OCCLUDED, SO
THERE CAN BE NOT FORMATION OF A
DIMER FORM OF THE PROTEIN.
SO THIS INDICATES THAT OUR
STRUCTURES TRULY REPRESENT AN
INHIBITORY STATE OF THE PROTEIN,
AND IT IS AN OUTER STATE BECAUSE
THEY ARE ALL N TERMINAL
INTERACTIONS THAT ARE HELPING TO
MAINTAIN THIS CONFORMATION.
SO VERY IMPORTANT CHARACTERISTIC
OF OUR STRUCTURE IS THAT WE WERE
ABLE TO SOLVE THE POSITION OF
THE LAST BINDING DOMAIN.
SO LAST BINDING DOMAIN HERE
SHOWN IN ORANGE, AND AS YOU CAN
SEE HERE, THE RAS BINDING DOMAIN
SITS ADJACENT TO THE KINASE
DOMAIN, AND ON TOP OF ONE OF THE
14-3-3PROTOMERS, AND THEY FORM
KIND OF AN EXTENSIVE INTERACTION
WITH AN AREA OF AROUND
400 AMSTRONS AND COMPLEMENT TEAR
RI BETWEEN THE BINDING DOMAIN
AND THE 14-3-3.
IF WE LOOK CLOSER TO THIS
INTERFACE, WE CAN SEE THAT THE
ALPHA-1 IN THE RAS BINDING
DOMAIN THAT IS ESTABLISHING SOME
CONTACTS WITH THE ALPHA 8, ALPHA
9 AND LOOP 8 IN 14-3-3.
SPECIFICALLY, WHEN WE LOOK AT
THE RESIDUES THAT ARE LOCATED,
THE 14-3-3 SITE OF THE
INTERFACE, WE CAN SEE THAT ALL
THE RESIDUES THAT ARE AT THIS
INTERFACE ARE CONSERVED BETWEEN
THE DIFFERENT ISOFORMS OF THE
14-3-3 FAMILY.
AS YOU MAY KNOW, 14-3-3 HAS
SEVEN DIFFERENT ISOFORMS BUT ALL
THE RESIDUES IN THIS INTERFACE
ARE CONSERVED BETWEEN THESE
DIFFERENT 7 ISOFORMS, SO THIS
INDICATES THAT REGARDLESS OF THE
COMPOSITION OF THE 14-3-3 CAN,
THIS INTERFACE CAN BE
ESTABLISHED.
THAT'S IMPORTANT BECAUSE THE
14-3-3 TENDS ON DIMER EYES, SO
YOU CAN HAVE THE -- DIMERIZE, SO
YOU CAN HAVE THE COMPLEX
DIFFERENT 14-3-3.
HOWEVER, WHEN WE LOOK AT THE RAS
BINDING DOMAIN RESIDUES WE SEE
THEY ARE NOT FULLY CONSERVED IN
THE FAMILY OF THE RAF PROTEINS,
SPECIFICALLY THERE ARE THESE TWO
MECHANISMS, 187 AND 186 IN THE
BRAF THAT THEY ARE DIFFERENT
FROM CRAF OR ARAF.
SO THESE TWO RESIDUESES, 186 AND
187 WE CAN SEE THEY CAN HAVE
MULTIPLE POTENTIAL INTERACTIONS
WITH 14-3-3.
SO EVEN THOUGH WE KNOW THAT THE
CRT HERE IN VIOLET IS KIND OF
LIKE THE KEY COMPONENT OF THE
OUTER HELIX BECAUSE IT MAKES
INTERACTIONS WITH THE KINASE
DOMAIN AND THE 14-3-3, WE WANTED
TO INVESTIGATE IF THE RAS
BINDING DOMAIN OF RBD ORANGE
ALSO DROIBTD THE MAINTAINING OF
THIS OUTER HELIX STATE BY
INTERACT WITH 14-3-3.
SPECIFICALLY WE WANTED TO SEE IF
THESE TWO RESIDUES 186 AND 187
WERE IMPORTANT FOR MAINTAINING
THIS OUTER STATE.
SO IN ORDER TO DO THAT, WE TOOK
MUTATIONAL APPROACH IN WHICH WE
MUTATE THESE TWO RESIDUES TO
FIRST THE RESIDUES AND TO OTHER
MEMBERS.
FAMILY.
-- MEMBERS OF THE FAMILY,
WHETHER IT'S PRESENT THIS ARAF
OR CRAF AND ALSO TO A SMALLER
AMINO ACID, LARGER ACIDS OR
TENDED TO CHARGE RESIDUES, AND
WE WANTED TO SEE IF BY DOING
THESE MUTATIONS BY MODULATING
THIS INTERFACE WE COULD SEE A
DIFFERENCE IN OUTER INHIBITION.
SO WITHOUT GOING INTO TOO MUCH
DETAILS ON THE ASSAY, WHAT WE DO
IS THAT WE USE A ACID THAT
ALLOWS US TO SEE WHAT WAS THE
DEGREE OF OUTER INHIBITION IN
THE PROTEIN.
WE ALSO USED A FOCUS FORM, HOW
ACTIVE THE CELLS ARE BASED ON
THE GROWTH OF THE CELLS.
BASICALLY WHAT WE CAN SEE HERE
IS THAT MOTD LAITD RESIDUES AT
THIS INTERFACE TRANSLATED IN
MODULATION IN OUTER INHIBITION
AND ACTIVITY OF THE PROTEIN, AND
THAT ALSO CORRELATED WITH THE
LEVELS OF APPROXIMATE PHOSFORMIC
IN THE CELLS, REMEMBER THAT MEK
IS THE SUBSTRATE FOR RAF.
SO ALL OF THIS INDICATED THAT
EVEN THE CRT'S, THE KEY
COMPONENT TO MAINTAIN THE OUTER
INHIBITORY STATE WE CAN SAY THAT
THE RAS BINDING DOMAIN ALSO
DROIBTD THE MAINTENANCE OF THE
OUTER INHIBITORY STATE OF THE
PROTEIN.
BUT OBVIOUSLY THE -- BUT OBVIOUN
OF THE BINDING DOMAIN IS TO BIND
TO RAS SO WE WANTED TO SEE WHAT
HAPPENS IF WE ADD RAS TO IT
COMPLEX AND IF THAT CAN GIVE US
SOME INDICATION OF HOW THIS WILL
HAVE TO TRANSITION FROM THE
MONOMER TO THE DIMER STATE.
SO AS YOU CAN SEE HERE, WE HAVE
THE STRUCTURE THAT WE SAW FOR
THE OUTER INHIBITORY STATE AND
WE KIND OF MODEL THE BINDING OF
THE RAS PROTEIN INTO THIS
MONOMER STRUCTURE.
SO WE DID THIS MODEL, BASICALLY
USING FOUR RESIDUE THAT IS ARE
PRESENT IN THE RAS BINDING
DOMAIN THAT THEY FORM IONIC
INTERACTIONS WITH RAS.
SO THESE ARE VERY WELL KNOWN
ESTABLISHED AND STUDIED
INTERACTIONS BETWEEN THE TWO
PROTEINS.
SO WE USE THIS MODULATOR BINDING
OF RAS TO RAF, AND WE ALSO
WANTED TO SEE IF OUR ISOLATED
OUTER COMPLEX WERE ABLE TO BIND
TO RAS.
SO WE DID ASSAYS AND WE CAN
DETERMINE THAT OUR OUTER
INHIBITORY COMPLEXES INTERACTS
WITH HIGH AFFINITY WITH PURIFIED
RAS PROTEIN.
WE ALSO DID SOME EXPERIMENTS
WHERE OUR ALPHA HELIX COMPLEX
WERE ABLE TO PULL DOWN R AS --
K-RAS AND WE CAN ABOLISH
INTERACTION BETWEEN RAF AND RAS.
SO I'M GOING BACK TO THE
MODELING OF THE BINDING, WHEN WE
MODELED THE K-RAS BINDING TO THE
RAS BINDING DOMAIN, WHAT WE CAN
SEE HERE IS THAT WHEN THE
INTERACTION BETWEEN KRAS AND RPD
IS ESTABLISHED, THERE IS GOING
TO BE -- RBD IS ESTABLISHED,
THERE WILL BE A CLASHING BETWEEN
THE KRAS AND 14-3-3 THAT IS
BINDING TO THE RAS BINDING
DOMAIN AND THERE WILL ALSO BE AN
ELECTROSTATIC REPULSION BETWEEN
THE KRAS AND THE 14-3-3 BECAUSE
THEY ARE BOTH NEGATIVELY
CHARGED.
SO BASICALLY, WHAT THIS TELLS US
IS THAT WHEN RAS BINDS TO THE
OUTER HELIX, THERE WILL BE RAF,
THERE NEEDS TO BE ORGANIZATION
OF THE 14-3-3 BINDING IN ORDER
FOR RAS COMPLETELY BIND TO BRAF.
THERE WAS ANOTHER INTERESTING
OBSERVATION, WHEN WE DID THIS
MODELING OF THIS BINDING, IS
THAT WHEN RAS BIND TO THE RBD,
THESE TWO MECHANISMS THAT WE
OBSERVED BEFORE WERE IMPORTANT
FOR THE BINDING BETWEEN THE RAS
BINDING DOMAIN AND THE 14-3-3,
THEY COME IN CLOSE PROXIMITY TO
RAS SO WE WANTED TO INVESTIGATE
IF THESE TWO RESIDUES WERE ALSO
INVOLVED TO THE RAS BINDING ONCE
THE 14-3-3 IS MOVED OUT OF THE
WAY.
IN ORDER TO DO THAT, WE PERFORM
ANOTHER TYPE OF ASSAY THAT
ALLOWS US TO STUDY INTERACTION
BETWEEN RAS PROTEIN AND BRAF
PROTEIN AND WITH A SERIES OF
MUTATIONS OF THESE TWO RESIDUE
AND IS WE SAW IF THERE WAS AN
EFFECT BETWEEN THE BINDING OF
RAS AND RAF AND IN FACT WE SEE
THAT AGAIN WHEN WE MODULATE
THESE TWO MECHANISMS, WE SEE A
DIFFERENT DEGREE OF BINDING
BETWEEN RAS AND RAF INDICATING
THAT THESE TWO RESIDUES IN FACT
PARTICIPATE IN THE BINDING
BETWEEN THE TWO PROTEINS.
ALSO WITH ASSAYS PULL DOWN
EXPERIMENTS WHERE WE OBSERVE THE
SAME THING, THAT THESE TWO
RESIDUES ARE IMPORTANT FOR THE
BINDING OF THE TWO PROTEINS.
SO BASICALLY, WHAT THIS IS
TELLING US IS THAT THESE TWO
MECHANISMS IN THE ALPHA-1 HELIX
IN THE RAS BINDING DOMAIN HAVE
KIND OF A DUAL FUNCTION.
IN THE OUTER INHIBITORY STATE,
THEY BIND WITH 14-3-3 AND HELP
TO MAINTAIN THE OUTER INHIBITORY
STATE, BUT WHEN RAS COME AND
BINDS TO THE RAS PROTEIN AND THE
14-3-3 MOVES MOVES OUT OF THE
WAY, THERE'S ALSO INTERACTION
WITH THE RAS PROTEIN, MAKING
INTERACTION BETWEEN THE TWO
PROTEINS.
SO IF WE PULL ALL OF THIS
TOGETHER, WE KIND OF PROPOSE A
MODEL ON HOW THE ACTIVATION OF
THE BRAF PROTEINS CAN HAPPEN
AFTER THE RAS PROTEIN
SOUTHBOUND.
SO WE START WITH THE CELLS WHEN
THE TWO PROTEINS ARE IN INACT
INACTIVE, KRAS BOUND TO GDP, WE
HAVE BRAF FLEEN OUTER INHIBITORY
STATE MAINLY BETWEEN
INTERACTIONS BETWEEN N TERMINAL
REGULATORY AND C TERMINAL
CATALYTIC DOMAIN AND WITH
INTERACTIONS WITH 14-3-3.
ONCE RAS IS ACTIVATED THE
BINDING OF GDP IT IS GOING
RECRUIT AND BRING BRAF TO THE
PLASMA MEMBRANE, SPECIFICALLY
FOR THE INITIAL IONIC
INTERACTIONS BETWEEN KRAS AND
RBD.
WHEN THIS INTERACTION HAPPENS,
THERE IS GOING TO BE A CLASH AND
ELECTROSTATIC REPULSION BETWEEN
THE RAS AND THE 14-3-3.
SO THIS 14-3-3 HAS TO BE
REORGANIZED.
WHEN THAT HAPPENS, IT ALLOWS FOR
THE KINASE DOMAIN TO REORGANIZE
AND FORM THE DIMER, THE RESIDUE
THAT FORM THE DIMER INTERFACE
BECOME EXPOSED AND THEY CAN'T
OBVIOUSLY REORGANIZE AND FORM
THE DIMER.
AND THEN THE PROTEIN BECOMES
ACTIVE.
SO THAT'S THE MODEL WE PROPOSED.
NOW MOVING FORWARD WHERE WE'RE
TRYING TO UNDERSTAND REGULATORY
OUTER INHIB -- INFIN HI BASE IS
SHARED BY OTHER MEMBERS OF THE
FAMILY, ESPECIALLY IF IT HAS
SAME OUTER FORMATION OF BRAF AND
PARTICULARLY IF THE DIMER
FORMATION HAPPENS IN THE SAME
WAY.
THAT'S WHAT WE ARE TRYING
INVESTIGATE NOW.
SO WE BASICALLY COME TO THE END
OF MY PRESENTATION.
I WANT TO THANK ALL THE MEMBERS
OF MY GROUP, PRINCIPALLY MY PI
WHICH IS DR. PING ZING,
DR. MORRISON AND DAVID IN THE
LABORATORY OF CELL AND
DEVELOPMENTAL BIOLOGY AND ALL
THE IF RESOURCES, AND ALSO OUR
CRYO-EM CENTER AND BIOWULF FOR
ALL THE COMPUTATIONAL RESOURCES
THAT ALLOW US TO STUDY THE
STRUCTURE OF THE PROTEINS, AND
THE CENTERS FOR BIOSTRUCTURAL
PHYSICS RESOURCES FOR ALL THE
RESOURCES FOR THE
CHARACTERIZATION OF THE COMPLEX,
AND ALL OF YOU FOR YOUR
ATTENTION.
[APPLAUSE]
>> (AWAY FROM MICROPHONE).
>> SO ALL THE GROWTH FACTOR
SIGNALS THAT INITIATE THE
CASCADE OF THE SIGNALLING
PATHWAYS FROM THE TRANSITION OF
KRAS FROM GTB TO GDP BINDING
STATE SO ONCE THE PROTEIN IS IN
THE GDP STATE IT CAN TBIEND THE
RAF PROTEINS, OTHERWISE THEY
WON'T BIND.
OKAY?
>> (AWAY FROM MICROPHONE).
>> YEAH, ALL THE DOMAINS WE CAN
HAVE A STARTING POINT BASICALLY
FROM IT CHRYSALIS STRUCTURES, SO
LIKE THE RAS DOMAIN, KINASE
DOMAIN, 14-3-3 PROTEINS THEY
WERE ALL DOMAINS THAT HAVE
ALREADY BEEN DEPOSITED IN THE
PPD SO WE DRINTD IS TO DO FROM
SCRATCH MODELING OF THE
STRUCTURES.
DISAISH ON WE DIDN'T HAVE -- SO
WE DIDN'T HAVE TO DO SCRATCH
MODELING OF THE STRUCTURES.
THANKS.
>> (AWAY FROM MICROPHONE).
>> SO YEAH, TR AT THAT POINT WE
DIDN'T HAVE ANY STRUCTURE OF THE
COMPLEX OF RAS BOUND TO BRAF.
I THINK LIKE THIS YEAR THERE WAS
A RELEASE OF A STRUCTURE, BUT
LOW RESOLUTION OF RAS BINDING TO
BRAF.
BUT AT THAT POINT, THERE WAS NOT
A STRUCTURE OF THE COMPLEX, SO
BASICALLY WHAT WE USED IS THAT F
WE LOOK AT HERE, THERE WAS KNOWN
INTERACTION OF THE RAS WITH JUST
THE R AF BINDING DOMAIN OF THE
PROTEIN WITH RAS AND THERE WAS A
CHRYSALIS STRUCTURE OF THIS TWO
DOMAIN BINDING BUT THAT WAS NOT
IN COMPLEX OF THE FULL LENGTH
BRAF.
JUST RAF BINDING DOMAIN YOU BIND
WITH THE RAS.
WE USE THAT HAD TO KIND OF
SUPERIMPOSE ON OUR COMPLEX.
THAT'S HOW WE MODEL WHAT WILL
HAPPEN WHEN THIS INTERACTION IS
ESTABLISHED.
>> (AWAY FROM MICROPHONE).
DO YOU HAVE ANY THOUGHTS ON
WHETHER OR HOW THIS METHOD COULD
BE USED, OR --
>> YEAH, I THINK THEY'RE
DEFINITELY VERY COMPLEMENTARY TO
EACH OTHER.
WHEN YOU LOOK AT SPECIFICALLY IN
OUR LAB, WE WORK A LOT WITH
KINASES.
SO IF YOU LOOK AT ALL THE
DEPOSIT, LIKE STRUCTURE FOR
INSTANCE IN ALPHAFOLD, THERE ARE
ALL MODELS OF KINASE ACTIVE
STRUCTURES.
SO IF YOU GO ON THERE AND YOU
LOOK AT THEM, YOU CAN HAVE A
STARTING POINT FOR WHAT YOU WANT
TO INVESTIGATE IN YOUR PROTEINS,
BUT WE HAVE SEEN HOW THESE
ACTIVE CONFORMATIONS THAT ARE
DEPOSITS, THEY DON'T -- WHEN YOU
DON'T HAVE ANY OTHER
INFORMATION, THEY ARE A GOOD
STARTING POINT FOR YOU TO KIND
OF FORMULATE YOUR HYPOTHESIS AND
START DOING YOUR EXPERIMENTS.
BUT DEFINITELY I FEEL LIKE WHEN
YOU DO THE STRUCTURE
DETERMINATION, YOU CAN GET LIKE
REALLY INSILENT OF HOW THE
PROTEIN IS REALLY LIKE WORKING.
SO SOMETIMES THEY WILL AGREE,
SOMETIME THEY WON'T.
YEAH.
THANKS.
>> (AWAY FROM MICROPHONE).
>> SO IF YOU LOOK AT JUST THE
KINASE ACTIVITY, THEY ARE VERY
SIMILAR IN TERMS OF THE
SUBSTRATES THAT THEY
PHOSPHORYLATE ARE ALSO VERY
SIMILAR.
THE SEQUENCE IDENTITY THEY HAIR
IS ALSO VERY HIGH, BUT IN
DISEASE STATES, YOU CAN SEE
DIFFERENT KIND OF -- THERE IS A
DIFFERENT FUNCTION, YOU CAN SEE
IN SOME TYPE OF CANCERS, THERE
ARE NO MUTATIONS IN BRAF AND
OTHER ONES THERE ARE NO
MUTATIONS IN CRAF.
THAT'S WHY WE ARE TRYING TO
UNDERSTAND THE STRUCTURE IF
THESE PROTEINS DIFFER IN THE
FORMATION SPECIFICALLY OF THESE
OUTER INHIBITORY COMPLEX ASK
ACTIVE COME PLEKS.
RIGHT NOW WE FINISHED THIS WHICH
WAS THE STRUCTURE OF THE BRAF
AND THEN WE MOVE IT TO TRY TO
STUDY THE CRAF PROTEINS, AND WE
HAVEN'T BEEN ABLE TO SOLVE YET
THE STRUCTURES, BUT WE HAVE SOME
PRELIMINARY DATA THAT INDICATES
THAT THIS OUTER INHIBITORY STATE
IS DIFFERENT.
THE MAIN DIFFERENCE WE ARE
SEEING SO FAR IS THAT THE CRT
DOMAIN THE ONE I SHOWED IN
VIOLET THAT IS IN THE ERNT OF
THE STATE IN THE BRAF MIGHT NOT
BE IN THE SAME POSITION IN THE
CRAF.
IF THAT IS THE CASE, IT MAY
INDICATE THAT A CRAF MAY HAVE A
MORE ACTIVE STATE BECAUSE IT HAS
A LESS OUTER STATE THAN BRA --
BECAUSE IT HAS ALPHA HELIX STATE
OF BRAF BUT THAT'S SOMETHING WE
ARE LOOKING AT NOW.
>> (AWAY FROM MICROPHONE).
>> SO BASICALLY, WHEN YOU
ANALYZE THE DATA, BASICALLY THE
PROCESS OF RECONSTRUCTING IS
LIKE AVERAGING, DOIRCHT THE
CLASSES -- DIFFERENT 2D CLASSES.
SO IF YOU HAVE SOME PORTIONS OF
THE PROTEIN, THIS IS A VERY
SIMPLIFIED OF THE PROCESS BUT
BASICALLY WHAT YOU USE.
SO IF YOU HAVE THE PROTEINS THAT
ARE VERY FLEXIBLE YOU CAN REALLY
POPULATE ONE STATE.
SO IT'S KIND OF LIKE YOU HAVE
SOMETHING HERE, SOMETHING HERE,
SOMETHING HERE.
SO THE AVERAGE THAT YOU GET IS
NOT VERY WELL DEFINED BECAUSE IT
IS ACTUALLY EVIDENT MOST
CONFORMATIONS SO IT MAKES
STRUCTURE TO HAVE LESS
RESOLUTION.
>> (AWAY FROM MICROPHONE).
>> YES.
>> SO ALL OF THIS IS DMURG
TWO-DIMENSIONAL -- IS OCCURRING
IN TWO-DIMENSIONAL.
YOU WERE SAYING THE N TERMINAL
IS THAT BECAUSE THEY INTERACT
WITH --
>> WHEN WE SAW THE STRUCTURES
THEY WERE IN SOLUTION AND WE
DON'T HAVE ANY MEMBRANE PRESENT,
BUT OBVIOUSLY WHAT WE'RE SUSPECT
SG THAT ALL OF THIS PROCESS
HAPPENS AT THE MEMBRANE BECAUSE
RAS IS LOCALIZED IN THE
MEMBRANE.
SO THERE IS ACTUALLY SOME
EVIDENCE THAT BRAF ITSELF MAY BE
INTERACTING WITH THE MEMBRANE BY
THE CRD DOMAIN.
BUT IN THE CYTOSOL, THE CRT WAS
COMPLETELY OCCLUDED BY THE
14-3-3 AND INTERACTIONS WITH THE
KINASE DOMAIN.
SO IT IS EXPECTED AT SOME POINT
DURING THE ACTIVATION THAT THE
CRD DOMAIN BECOMES EXPOSED AND
ENTER ABLGHTS ALSO WITH THE
MEMBRANE -- INTERACTS ALSO WITH
THE MEMBRANE BECAUSE THERE IS
SOME EVIDENCE OF THAT HAPPENING.
SO I GUESS YES TO FULLY
UNDERSTAND THIS ACTIVATION
MECHANISM, IT WOULD PROBABLY BE
VERY IMPORTANT TO TRY TO
UNDERSTAND HOW THIS HAPPENS IN
THE CONTEXT OF SOME KIND OF
MEMBRANE, SO PROBABLY SOME
LIPIDS, AS A LOT OF ACTIVE
RESEARCH IN THAT AREA.
THANK YOU VERY MUCH.
[APPLAUSE]
[THE EVENT CONCLUDED AT
11:42 A.M. EST]