>> TODAY'S SESSION OF THE
WEDNESDAY AFTERNOON LECTURE
SERIES HERE AT THE NIH FOR THOSE
WHO DON'T KNOW ME, I'M HEAD OF
THE SECTION OF STRUCTURAL AND
CHEMICAL BIOLOGY OF MEMBRANE
PROTEINS IN THE INTRAMURAL
PROGRAM OF NICHD.
TODAY'S TALK IS HOSTED BY THE
STRUCTURAL BIOLOGY INTEREST
GROUP OF WHICH I'M ONE OF THE
COORDINATORS, AND IT'S A REAL
PRIVILEGE AND HONOR FOR ME TO
INTRODUCE TODAY'S SPEAKER,
PROFESSOR JOHN KURIYAN, WHO
COMES TO US FROM THE UNIVERSITY
OF CALIFORNIA AT BERKLEY,
CHANCELLOR'S PROFESSOR IN THE
DEPARTMENTS OF MOLECULAR AND
CELL BIOLOGY AND THE DEPARTMENTS
OF CHEMISTRY AS WELL AS A HOWARD
HUGHES MEDICAL INSTITUTE
INVESTIGATOR.
JOHN WAS BORN AND RAISED IN
INDIA AND RECEIVED HIS
UNDERGRADUATE DEGREE IN
CHEMISTRY FROM PENNSYLVANIA,
THEAND
THEN HE WENT TO THE DEPARTMENT
OF CHEMISTRY AT HARVARD WHERE HE
GOT HIS PH.D. IN PHYSICAL
CHEMISTRY FOLLOWED BY A VERY
SHORT POSTDOCTORAL POSITION
WHERE HE WAS .
JOHN'S THESIS RESEARCH WAS
MOSTLY COMPUTATIONAL CHEMISTRY
FOR THOSE OF YOU WHO ARE SEEN AS
USERS, YOU REMEMBER THE
REFINEMENT OF R FACTOR.
SO WHEN HE STARTED HIS LAB AT
THE ROCKEFELLER UNIVERSITY AND
MADE THE SWITCH TO EXPERIMENTAL
STRUCTURAL BIOLOGY, IT WAS A
DRASTIC AND BOLD MOVE, AND TO
JOHN'S KRE IT,
JOHN'S CREDIT, HE DID NOT ONLY
SURVIVE THE TRANSITION AS WELL
AS ANYONE COULD IMAGINE, HIS
RESEARCH HAS THRIVED ALONG TWO
VERY DISTINCT DIRECTIONS, SIGNAL
TRANSDUCTION BY PROTEIN KINASES.
JOHN ROSE THROUGH THE RANKS AT
ROCKEFELLER AND BECAME THE
HAGGERTY PROFESSOR.
IN 2001, JOHN MOVED TO UC
BERKLEY, WHERE HE HAS REMAINED
SINCE THEN.
JOHN'S HONORS AND ACCOLADES HAVE
BEEN MANY.
HE'S A MEMBER OF BOTH THE
NATIONAL ACADEMY OF SCIENCES AND
MEDICINE.
HE HAS RECEIVED MANY AWARDS
INCLUDING THE PRESTIGIOUS STEIN
AND MOORE AWARD FROM THE PROTEIN
SOCIETY, THE MARK AWARD FROM THE
AMERICAN SOCIETY OF BIOCHEMISTRY
AND MOLECULAR BIOLOGY, AND THE
RICHARD LANCEBURY AWARD FROM THE
NATIONAL ACADEMY OF SCIENCES AND
MANY OTHERS I DON'T HAVE TIME TO
NAME HERE.
HOWEVER ANOTHER SIGNIFICANT MARK
OF AN ACADEMIC SCIENTIST IS THE
SCIENTIST THAT THEY TRAIN AND
JOHN HAS TRAINED AN OUTSTANDING
GROUP OF SCIENTISTS WHO HAVE
GONE ON TO BECOME LEADERS IN
THEIR OWN RITE.
FOR THOSE WATCHING REMOTELY, WE
ENCOURAGE YOU, YOU CAN
PARTICIPATE IN THE Q & A AFTER
TODAY'S PRESENTATION.
YOU CAN DO SO BY CLICKING ON THE
BUTTON JUST BELOW YOUR VIDEOCAST
WINDOW THAT SAYS "SEND LIVE
FEEDBACK."
CLICK ON THAT, TYPE IN YOUR NAME
AND QUESTION, AND WE WILL RELAY
THE QUESTION TO DR. KURIYAN AT
THE END OF THE LECTURE.
YOU CAN ALSO SUBMIT A QUESTION
AT ANY TIME AND DON'T HAVE TO
WAIT UNTIL THE END.
FOR THOSE OF YOU IN THE ROOM
TODAY, PLEASE USE THE MICS ON
EITHER SIDE.
ALSO WE ARE OFFERING CONTINUING
MEDICAL EDUCATION CREDITS FOR
THIS PRESENTATION.
THE CME CODE FOR TO THE IS
37946.
THE TITLE OF JOHN'S TALK IS DEEP
MUTAGENESIS ANALYSIS OF
EVOLVABILITY IN A DNA POLYMERASE
AAA PLUS CLAMP LOADER SYSTEM.
JOHN, THANKS FOR ACCEPTING OUR
INVITATION.
[APPLAUSE]
>> THANK YOU VERY MUCH FOR THE
PRIVILEGE OF SPEAKING TO YOU
THIS AFTERNOON.
IN THIS HYBRID
FORMAT.
IT'S NICE TO SEE PEOPLE ALIVE
AND WELL IN THE AUDIENCE BECAUSE
IF YOU CAN MAKE EYE CONTACT WITH
ONE OR TWO, IT LETS YOU KNOW
THAT YOU'RE NOT SPEAKING INTO
THE VOID.
AS WE WOULD TEACH UNDERGRADUATES
OVER THE PANDEMIC.
I'M GOING TO TALK TO YOU TODAY
ABOUT SOME WORK WE'VE BEEN DOING
TO UNDERSTAND THE ABILITY OF A
MOLECULAR MACHINE KNOWN AS THE
DNA POLYMERASE CLAMP LOADER TO
TOLERATE ADDITIONAL INSULTS
WHILE IT'S FUNCTIONING IN THE
DNA REPLICATION SYSTEM.
SO WHILE DNA PO POLYMERASE SYSTEMS
ACHIEVE GREAT SPEED THROUGH
ATTACHMENT TO A SLIDING CLAMP,
AND THE SLIDING CLAMP IS A
CIRCULAR PROTEIN THAT GOES
AROUND DNA, AND BY ATTACHING THE
POLYMERASE TO THE SIDING CLAMP
THE SYSTEM CAN ACHIEVE VERY HIGH
SPEED WITHOUT LETTING GO OF DNA.
SO THIS IS A PRINCIPLE THAT
EMERGED, THE STRUCTURAL BASIS
FOR THIS EMERGED FROM STUDIES
DONE OVER THE YEARS BY MY LAB
AND THE LAB OF MICHAEL
O'DONNELL.
AND THIS SIMPLIFIED DIAGRAM
ESSENTIALLY SHOWS YOU HOW THE
DNA POLYMERASE CLAMP LOADER
SYSTEM WORKS TO LOAD SLIDING
CLAMPS ON TO DNA, AND IT DOES
THIS REPEATEDLY FOR EVERY
FRAGMENT THAT'S GENERATED.
SO THE DNA POLYMERASE SLIDING
CLAMP SHOWN HERE IS A COMPLEX OF
FIVE DIFFERENT PROTEINS LABELED
A, B, C, D, E, AND THEY ARE ATP
ACES OF THE TRIPLE A FAMILY AND
THE ACTION OF THE MOLECULAR
MACHINE IS BASED ON THE ABILITY
TO HYDROIZE ATP.
I'M GOING TO BE TELLING YOU
TODAY ABOUT ONE SPECIFIC CLAMP
LOADER CALLED THE T4 PHAGE CLAMP
LOADER SYSTEM, AND IN THAT
PARTICULAR ONE, THERE ARE TWO
GENES THAT ENCODE THE FIVE
PROTEIN COMPLEX, ONE GENE
ENCODES THE PROTEIN COLORED
GREEN HERE, WHICH IS ACTUALLY A
DEGENERATE AAA PLUS PROTEIN SO
IT DOESN'T LOOK LIKE THE OTHERS
BUT IT ORIGINATED LONG AGO FROM
THE OTHERS.
AND THE OTHER GENE ENCODES FOUR
OF THE ATP ACES WHICH ARE
LABELED A, B, C, D AND E IN THIS
COMPLEX SO TOGETHER THEY FORM AN
ASSEMBLY THAT DOES NOT HYDROLIZ,
ATP UNTIL IT RECOGNIZES THE
SLIDING CLAMP AND IT RECOGNIZES
A PRIMARY JUNCTION SHOWN
SCHEMATICALLY HERE.
WHEN THOSE TWO THINGS ARE
RECOGNIZED, THE ASSEMBLY
HYDROLYZES ATP WHICH RELEASES
THE CLAMP LOADER FROM THE
SLIDING CLAMP AND DNA, LEAVING
THE SLIDING CLAMP ON DNA.
SO THAT'S THE ESSENTIAL FUNCTION
OF A CLAMP LOADER.
THE STRUCTURE EV THESE THAT TOLD
MANY OF THE ESSENTIAL ASPECTS OF
HOW THESE THINGS PULL TOGETHER
AND HOW THEY WORK BUT TODAY I
WANT TO FOCUS TWO QUESTIONS THAT
CAME OUT OF THINGS THAT WE WERE
WONDERING ABOUT.
THE FIRST QUELL IS WE KNOW THAT
THE DIVERSITY OF AAA+ PROTEINS,
CLAMP LOADERS SPECIFICALLY,
AAA -- IS ENORMOUS BOTH IN
SEQUENCE AND STRUCTURAL DETAILS.
LIKE ALL BIOLOGISTS WHO STUDY
MOLECULAR SYSTEMS AND EVEN KNOW
CROWSCOPIC SYSTEMS WE WONDER HOW
DOES NATURE COME UP WITH THIS
DIVERSITY AND STRUCTURE, WHEN
MAINTAINING AN ESSENTIAL
MECHANISM?
ONE WAY TO ASK THAT IS TO ASK
THE QUESTION, WHAT'S THE
CAPACITY OF THE CLAMP LOADER
SYSTEM TO ACCEPT CHANGES TO ITS
SEQUENCE WHILE IT ACTUALLY
PERFORMING A CRITICAL TASK.
IN THIS CASE, DNA REPLICATION.
SO WHAT I'M GOING TO TELL YOU IS
ABOUT A SYSTEM DEVELOPED IN MY
LAB WHICH ALLOWS US TO PROBE THE
MUTATIONAL TOLERANCE OF THE
SYSTEM WHILE IT'S ACTUALLY
REPLICATING DNA.
AND THE SECOND QUESTION WE'D
LIKE TO ASK AND ANSWER IS WHAT
DOES THE MUTATIONAL RESPONSE OF
THE SYSTEM TELL US ABOUT THE
MECHANISM?
NOW WE KNOW A LOT ABOUT THE
MECHANISM FROM THE STRUCTURAL
WORK AND THE BIOCHEMISTRY THAT
WE'VE DONE, BUT AS YOU'LL SEE,
THE ANALYSIS OF THE MUTATIONAL
RESPONSE DID TEACH US SOME
COMPLETELY UNEXPECTED THINGS
ABOUT THE SYSTEM.
SO THAT'S WHAT THIS LECTURE IS
GOING TO BE ABOUT.
SO THE WAY THE EXPERIMENTS WERE
CARRIED OUT WAS TO USE AN
EXPERIMENTAL PLATFORM DEVELOPED
WHICH IS EXTREMELY ROBUST IN
TERMS OF BEING ABLE TO TELL US
THE EFFECT ON FITNESS OF ANY
MUTATION MADE IN THE CLAMP
LOADER SYSTEM.
SO FIRST I'D LIKE TO JUST
EXPLAIN WHAT THIS PLATFORM IS
USING THIS DIAGRAM, THEN I'D
LIKE TO SHOW YOU SOME DATA WHICH
CONVINCED US THAT THE SYSTEM IS
ACTUALLY REALLY ROBUST IN TERMS
OF TELLING US THE RELATIONSHIP
BETWEEN SEQUENCE AND THE
FUNCTION OF THE PROTEIN.
SO AS I MENTIONED, THE SYSTEM
WE'RE USING IS A MODEL SYSTEM IS
T4 BACTERIOPHAGE WHICH IS
SPECIAL BECAUSE T4 BACTERIOPHAGE
BRINGS WITH IT ITS OWN DNA
REPLICATION PROTEINS WHETHER
IT -- E. COLI.
SO IT BRINGS A SLIDING CLAMP,
CLAMP LOADER, DNA LIGASE,
STRANDING DNA BINDING PROTEIN,
ALL OF WHICH IT USES INSTEAD OF
USING THE E. COLI PROTEINS.
IN ADDITION, THE -- AND THIS IS
NOT REALLY IMPORTANT FOR THE
WORK I'M GOING TO TELL YOU ABOUT
TODAY BUT IN ADDITION, THESE
PROTEINS THAT THE T4 SYSTEM
BRINGS WITH IT ARE MORE SIMILAR
TO THE EUKARYOTIC REPLICATION
PROTEINS, THEY'RE ACTUALLY A
MUCH BETTER MODEL FOR EUKARYOTIC
REPLICATION PROTEINS THAN IS THE
BACK
BACTERIAL SYSTEM.
SO WHAT SUBU DID WAS -- THE
SPECIFIC REPLICATION PROTEINS OF
INTEREST COULD BE ANY OF THE
REPLICATION PROTEINS BUT TODAY
I'M GOING TO TALK ABOUT THE
SLIDING CLAMP AND THE CLAMP
LOADER, ARE DELETED FROM THE
T4 BACTERIOPHAGE SO THOSE SHOWN
IN GREY DON'T HAVE THE
REPLICATION PROTEINS, THE
SLIDING CLAMP, THE CLAMP LOADER.
SO THEY CAN INFECT E. COLI BUT
THEY'RE UNABLE TO REPLICATE.
HOWEVER, IF THE E. COLI CONTAIN
PLASMIDS, SHOAP HERE AS THESE
COLORED CIRCLES THAT CONTAIN
CODING REGIONS FOR THESE
REPLICATION PROTEINS THEN THE
E. COLI WILL MAKE THE PROTEINS
AND THOSE PROTEINS CAN BE USED
BY THE T4 TO REPLICATION.
SO IF THE E. COLI CONTAINS A
LIBRARY THAT IS A VARIETY OF
VARIANTS OF THESE REPLICATION
PROTEINS THEN ONES THAT WORK
BETTER WILL PRODUCE MORE PHAGE
THAN ONES THAT DON'T WORK SO
WELL.
THERE'S ONE MORE THING THAT HAD
TO BE DONE IN ORDER TO SET UP A
SYSTEM WHERE WE COULD USE DEEP
SEQUENCING TO ACTUALLY COUNT THE
SUCCESS OF ANY VARIANT.
THAT WAS CRISPR/CAS 12 WORKING
IN REVERSE.
YOU KNOW THAT NORMALLY THE
CRISPR IS PULLING OUT DNA FROM
THE BACK BACTERIOPHAGE AND TESTING
IT, THAT'S THE CLASSICAL
MECHANISM OF PROTECTION THAT THE
CRISPR SYSTEM EMPLOYS, BUT HERE
THE CRISPR IS USED TO PUT THE
GENES -- SO WHEN A PHAGE INFECTS
A PARTICULAR E. COLI, THE CRISPR
SYSTEM WILL PUT THESE GENES BACK
INTO THE PHAGE THROUGH CRISPR
RECOMBINATION.
SO IF THESE GENES ARE ACTUALLY
SUCCESSFUL AT PRODUCING
BACTERIOPHAGE, THEN THE
BACTERIOPHAGE WILL LISE THE E
OWE
E. COLI, THE MORE OF THAT
VARIANT WILL BE PRODUCED IN
SOLUTION.
SO THEN THE EXPERIMENT IS
CONCEPTUALLY VERY SIMPLE.
YOU USE ILLUMINA DEEP SEQUENCING
OR ANY DEEP SEQUENCING METHOD TO
SEQUENCE THE LIBRARY THAT WENT
INTO THE EXPERIMENT THOUSANDS OF
DIFFERENT VARIANTS IN THE POT,
AND THEN YOU CAN BASICALLY
NORMALIZE THE DATA TO TELL YOU
WHICH VARIANTS WORK BETTER THAN
THE OTHERS.
AND YOU CAN ACTUALLY THEN TAKE
THESE PHAGE, IF YOU WISH, AND
USE THEM TO REINFECT E. COLI AND
SET UP A COMPETITION, IN THAT
CASE YOU'D BE DOING A NATURAL
EVOLUTION VARIATION OF THIS
EXPERIMENT.
SO THE ESSENTIAL IDEA BEHIND
THIS PLATFORM WAS PUBLISHED IN
2021 TOWARDS -- I FORGET WHEN
EXACTLY IT WAS PUBLISHED BUT
IT'S IN THE LITERATURE SO YOU
CAN READ ABOUT IF YOU'RE
INTERESTED, BUT IT REALLY DOES
PROVIDE US WITH A VERY ACCURATE
MEASUREMENT OF THE FITNESS OF A
VARIANT.
ACCURATE IN TERMS OF WHAT WE
KNOW ABOUT CHEMISTRY AND
BIOCHEMISTRY AND ACCURATE IN
TERMS OF REPRODUCIBILITY AND
BIOCHEMICAL VALIDATION.
SO TO GIVE YOU A SENSE OF THE
ROBUSTNESS OF THE PLATFORM BUT
ALSO TO EXPLAIN THE IMPORTANCE
OF A MOTIF IN THE CLAMP LOADER
THAT WILL BECOME CENTRAL TO SOME
OF THE EXPERIMENTS I'M GOING TO
END THIS LECTURE WITH, I WANT TO
INTRODUCE YOU FIRST TO A MOTIF
IN THESE ATPASES CALLED THE DEXD
MOTIF.
NOW ANYBODY WHO'S FAMILIAR WITH
RNA HELICASES WILL KNOW ABOUT
THE DEAD BOX HELI DID THE CASES.
THEY WERE DISCOVERED MAYBE IN
THE LATE EIGHTIES.
THEY'RE A GROUP OF RNA
HELICASES, VERY IMPORTANT AT
DIFFERENT ASPECTS OF BIOLOGY
THAT HAVE AT THE HEART ATA
PROTEIN A KNOW DEEF DEAD, HENCE,
DEAD BOX.
THIS PARTICULAR ATPASE, THE
T4 BACTERIOPHAGE CLAMP LOADER,
HAS DE15 LAL NEEN SO I WRITE
THAT AS XD.
INSTEAD OF ALANINE.
IN THE STRUCTURAL DIAGRAM, WHAT
YOU SEE IS WHERE THE DEAD BOX IS
LOCATED.
THE D AND THE E OF THE DEAD BOX,
ALL COLORED RED HERE, THE D AND
THE E ALSO CALLED A WALKER A
MOTIF BECAUSE JOHN WALKER
IDENTIFIED THE IMPORTANCE OF
THIS ON THE F1 ATPASE SYSTEM
THAT MAKES ATP.
SO THESE RESIDUES ARE ANCIENT
EVOLUTIONARILY, AND THEY'RE
CRUCIALLY IMPORTANT.
THEY'RE IN THE MACHINE THAT
MAKES ATP, THEY START OFF ALL
KINDS OF PROCESSING AFTER
TRANSCRIPTION.
WHAT THE D AND THE E DO IS THAT
THEY COORDINATE A MINE BELOW THE
ATP SHOWN HERE IN COLORS, AND
ACTIVATE A WATER MOLECULE WHICH
IS NOT SHOWN HERE.
SO THEY'RE CRUCIAL REGS RESIDUES AND
ALWAYS FOUND IN THIS FAMILY OF
ATPASES.
THE THIRD ACIDIC RESIDUE IS NOT
A CATALYTIC RESIDUE.
WHAT YOU SEE DOING HERE IS IT'S
COORDINATING AN ARGININE, WHICH
YOU SEE IN YELLOW AND BLUE.
THAT ARGININE, LABELED ARGININE
122, IS PRESENTED BY NEIGHBORING
SUBUNIT, NOT THIS ONE.
SO IT'S IMPORTANT FOR
INTERFACIAL COUPLING.
SO THAT'S AN INTRODUCTION TO THE
DEAD BOX MOTIF.
AND WHAT I WANT TO SHOW YOU HERE
ARE A FEW THINGS.
FIRST, LET'S LOOK AT THE LOWER
LEFT.
IN THE LOWER LEFT IS A SEQUENCE
LOGO.
AND THE WAY THE SEQUENCE LOGO IS
GENERATED BY SUBU WAS TO ALIGN
1,000 PHAGE GENOMES, THESE ARE
ALL BACTERIOPHAGE GENOMES OF
WHICH T4 IS ONE.
IT SHOWS YOU THE CONSERVATION OF
RESIDUES ACROSS THIS SEGMENT OF
1,000 PHAGES.
SO IT'S ACTUALLY A PRETTY
RESTRICTED LIBRARY.
IT ISN'T LOOKING AT THE BROADER
FAMILY OF ATPASES.
AND YOU'LL SEE THE DEAD BOX
MOTIF SPELLED OUT.
D, E, A, D.
THE FIRST THING TO NOTE IS THE
T4 BACTERIOPHAGE IS
IDIOSYNCRATIC.
IT DOESN'T HAVE THE A, IT HAS AN
F.
NOW, LET'S MOVE OVER TO THE HEAT
MAP IN THE MIDDLE HERE.
AND WHAT THIS HEAT MAP IS
SHOWING YOU IS A SEQUENCE THAT
CORRESPONDS TO THE DEAD BOX
REGION DEFDRSGLAE AS IS WRITTEN
ALONG THE HORIZONTAL AXIS.
AND AT EACH POSITION WITH THE
SATURATION MUTAGENESIS
EXPERIMENT USING THE
BACTERIOPHAGE PLATFORM SUBU
DEVELOPED DOES IS TO REPLACE
THAT RESIDUE WITH ONE OF THE 20
AMINO ACIDS OR STOP CODE STAR.
FOR EACH ONE OF THOSE
SUBSTITUTIONS, THE EFFECT ON
PHAGE HELP RICATION IS IS MEASURED AND SH OWN ON A
LOG 10 SCALE.
WHAT DARK BLUE TELLS YOU IS THAT
THE MUTATION LEADS TO LOSS OF
FUNCTION.
AND IF IT'S REALLY DARK BLUE, IT
LIKE AT LEAST 100 FOLD DOWN.
SO WHEN WE LOOK AT THIS, WE
START TO SEE THAT THINGS START
TO MAKE CHEMICAL SENSE.
THE D AND THE E, THE FIRST TWO
RESIDUES, CANNOT BE SUBSTITUTED
EVEN BY ANOTHER ACIDIC RESIDUE
WITHOUT TOTAL LOSS OF FUNCTION
AS MEASURED BY PHAGE PRODUCTION.
THAT MAKES SENSE BECAUSE IF
YOU'RE COORDINATING MAGNESIUM
AND ACTIVATING A WATER MOLECULE,
IF YOU SHIFT THE POSITION OF THE
CARBOXYL GROUP BY EVEN A
FRACTION OF AN ANGSTROM, YOU
LOSE ACTIVITY.
AND THE EXPERIMENT, THE DEEP
MUTAGENESIS EXPERIMENT IS
REFLECTING THAT CHEMICAL TRUTH.
IF YOU MOVE OVER AND LOOK AT THE
FOURTH POSITION, WHICH IS
ASPARTATE, YOU'LL SEE THAT'S
MORE -- SLIGHTLY MORE TOLERANT,
THE ASPARTATE CAN BE REPLACED BY
GLUTAMATE IN THE T4 SYSTEM.
AND AS I MENTIONED EARLIER, THAT
ASPARTATE IS NOT A CATALYTIC
RESIDUE, IT'S NOT INVOLVED IN
POSITIONING ANYTHING FOR
CATALYSIS OR INTERACTING WITH AN
ARGININE FOR INTERFACIAL
STABILIZATION.
WE'LL COME BACK TO THAT AND LOOK
AT IT IN MORE DETAIL LATER, BUT
YOU CAN UNDERSTAND THAT MAYBE
ASPEN GLUE WOULD STILL WORK.
AND IN FACT, IF YOU GO DOWN AND
LOOK OVER HERE, CYSTINE ALSO
WORKS.
AND WHEN YOU LOOK AT THIS LITTLE
BIT OF THE HEAT MAP SHOWING
MUTATIONAL RESPONSE, YOU START
TO LEARN THINGS THAT ARE VERY
INTERESTING.
I'M NOT GOING TO BELABOR THIS
MUCH, I JUST WANT TO TELL YOU
WHY THESE EXPERIMENTS -- SHOW
YOU WHY THESE EXPERIMENTS ARE SO
INTERESTING TO US.
SO FIRST OF ALL, THESE ARE DEAD
BOX PROTEINS.
BUT T4 DOESN'T HAVE A, IT HAS
PHENYL ALANINE IN THE THIRD
POSITION.
WHAT THIS EXPERIMENT HERE TELLS
US, IF YOU DRAW YOUR EYE TO THE
PURPLE BOX, WHAT IT TELLS US IS
THAT IF YOU ACTUALLY PUT A INTO
THE T4 CLAMP LOADER, YOU GET
LOSS OF FUNCTION.
SO AGAIN, IT'S TELLING US THAT
THIS PARTICULAR PROTEIN IS
IDIOSYNCRATIC IN ITS SEQUENCE IT
USES.
IT DOESN'T AT CERTAIN POSITIONS
MATCH THE CONSENSUS.
MOST INTERESTINGLY IF YOU MOVE
OVER AND LOOK AT THIS ARGININE
111, YOU'LL SEE THIS ARGININE
RESIDUE CANNOT BE REPLACED BY
ANYTHING, NOT EVEN LYSINE.
THE ONLY THING THAT WORKS IS
ARGININE.
AND THIS ARGININE IS FORMING AN
ION PAIR WITH A PHOSPHATE
BACKBONE OF DNA AND IT'S FORMING
THE CLASSIC ARGININE PHOSPHATE
BY DENTATE HYDROGEN BONDING
SYSTEM THAT WE SEE IN OUR
CRYSTAL STRUCTURE.
YOU CAN'T REPLACE IT WITH
ANYTHING, NOT EVEN LYSINE,
BECAUSE LYSINE WOULDN'T DO THAT.
BUT IF YOU LOOK AT THE SEQUENCE
LOGO WHICH TELLS YOU WHAT THE
POPULATION OF PHAGE CLAMP
LOADERS IS DOING, ARGININE ISN'T
EVEN SEEN IN THE LOGO AT THIS
POSITION.
SO WHAT THAT SAYS IS THAT THE
IDIOSYNCRASIES ARE BECOMING
EVIDENCE NOW.
ARGININE IS CRITICAL FOR
T4 CLAMP LOADER, MOST OF THE
PHAGE CLAMP LOADERS DON'T USE
ARGININE.
THEY'RE DOING SOMETHING ELSE.
THIS IS WHY THE DEEP MUTAGENESIS
EXPERIMENT IS ACTUALLY GIVING US
INFORMATION THAT'S ADDING ON TO
THE INFORMATION THAT THE
SEQUENCE ALIGNMENT IS TELLING
US.
AND THE REST OF THE TALK WILL
REALLY BE TRYING TO UNDERSTAND
WHAT'S THAT EXTRA INFORMATION
THAT IT'S TELLING US AT LEAST
OUR INITIAL GUESSES AS TO WHAT
THAT EXTRA INFORMATION IS ABOUT.
SO IF YOU LOOK AT WHERE THE MOST
SENSITIVE RESIDUES ARE, THE ONES
THAT YOU RILEY YOU REALLY CANNOT CHANGE T O
ONE OR TWO MORE OTHER RESIDUES,
YOU'LL FIND THESE RESIDUES
COLORED BLEUL IN THIS BLUE IN THIS RESIDU E
FORM A BELT AROUND THE DNA.
THIS BELT IS VERY SATISFYING TO
LOOK AT IN DETAIL.
IT CONNECTS EACH ATP SHOWN IN
RED TO DNA SHOWN IN THE CENTER
IN BLACK, CONNECTS THE DNA AND
THE ATP TO THE SLIDING CLAMP
WHICH IS SHOWN UNDERNEATH, A
WIRE DIAGRAM.
THIS IS A CRYSTAL STRUCTURE DONE
BY MY GROUP ABOUT 10 YEARS AGO.
SO THE MOST CRITICAL RESIDUES
ARE WHERE YOU EXPECT THEM TO BE.
THIS BINDS ATP COOPERATIVELY TO
THE DNA, TO THE SLIDING CLAMP.
THEY INTEGRATE ALL THAT
INFORMATION ALLOWING THE SYSTEM
TO FIRE ATP HYDROLYSIS ONLY WHEN
THIS IS CORRECTLY CONFIGURED.
BUT THE REALLY STARTLING THING
IN THIS DIAGRAM IS WHAT'S NOT
THERE.
EVERY SINGLE RESIDUE IN THIS
STRUCTURE, IN THE SLIDING CLAMP,
IN ALL SUBUNITS INCLUDING THE
ONE COLORED GREEN WERE MUTATED
IN SUBU'S EXPERIMENT.
BUT THEY'RE NOT COLORED BLUE.
AND WHAT THAT MEANS IS, ALL
THOSE RESIDUES THAT ARE NOT
COLORED, WHICH IS THE GREAT
MAJORITY OF THE STRUCTURE, CAN,
IN FACT, ACCEPT MANY, MANY
SUBSTITUTIONS AND STILL KEEP
WORKING.
SO WHAT THIS DIAGRAM ACTUALLY
REFLECTS IS SOMETHING WE ALREADY
KNOW FROM THE MUTAGENESIS
STUDIES OF SMALL PROTEINS THAT'S
REFLECTING THE AMAZING TOLERANCE
OF PROTEINS TO MUTATIONAL
INSULT.
BUT IT'S SAYING THAT EVEN FOR A
LARGE SYSTEM LIKE THIS, DOING
SOMETHING'S CRUCIAL AS
REPLICATING THE DNA, IT CAN
ACCEPT SINGLE POINT MUTATIONS.
SOME OF THEM QUITE STARTLINGLY
DIFFERENT FROM THE SEQUENCE
WHILE KEEPING ON WORKING.
NOW, WHEN WE LOOK AT THE
RESIDUES REALLY SENSITIVE TO
MUTATION, WE DID DISCOVER ONE
THING, WHICH IS ACTUALLY
IMPORTANT EVIDENCE IN THE
STRUCTURE BUT I WANT TO POINT
THIS OUT TO YOU JUST TO FINISH
WRAPPING UP THE RESULTS OF THE
FIRST ROUND OF EXPERIMENTS.
AND THAT IS THAT WE KNOW THE
SYSTEM IS HIGHLY COOPERATIVE
WHEN IT COMES TO DNA BINDING AND
ATP BINDING.
SO YOU'D EXPECT SINCE THERE ARE
FOUR ATP BINDING SITES AND THE
ATP IS COLORED RED HERE, YOU'D
EXPECT THE SYSTEM HAS ALLOSTERIC
COMMUNICATION THAT GOES FROM ONE
SITE TO THE OTHER, IT SURELY
DOES, AND TO DNA.
BUT WHAT YOU'LL SEAL IN SEE IN THIS VIEW
IS THERE'S REALLY NO
CONSERVATION OF SEQUENCE BETWEEN
EACH ATP SIE. WE NOW SITE.
WE NOW KNOW THAT'S BECAUSE
SECONDARY ELEMENTS PLAY A
CRITICAL ROLE IN THE
COMMUNICATIONS, WE'VE DONE
EXPERIMENTS TO ADDRESS THAT SO
YOU DON'T NEED A SPECIFIC
SEQUENCE, THERE'S ONLY ONE POINT
OF STRICT CONSERVATION
CONNECTING THE ATP BINDING SITES
AND THAT'S GLUTAMINE 118.
WE HADN'T NOTICED ITS IMPORTANCE
BEFORE.
IF YOU LOOK AT WHAT GLUTAMINE
118 IS DOING, IT'S A GLUTAMINE
SITE CHAIN THAT DOES WHAT
GLUTAMINE IS SO GOOD AT DOING.
GLUTAMINE CAN MIMIC -- IT GOT
THE CAPACITY TO MIMIC BACKBONE
HYDROGEN BONDING IN A BETA
SHEET.
SO WHAT IT'S ACTUALLY DOING IS
IT'S STAPLING THIS PURPLE HELIX
TO THIS YELLOW HELICAL STRUCTURE
AND LOOP HERE.
WHEN YOU SEE THIS, WE CALL THESE
THREE HELICES, YELLOW, MAGENTA
AND ORANGE, WE CALLED THEM THE
CENTRAL COUPLER TOGETHER BECAUSE
YOU'LL SEE WHAT'S HAPPENING IS
THIS IS THE POINT WHERE THE
CLAMP LOADER IS INTEGRATING ALL
THE INFORMATION THAT IT NEEDS TO
INTEGRATE.
IN RED HERE IS THE DEAD BOX, OR
DEXD OF THIS PARTICULAR VARIANT,
AS PAR TART, GLUTAMATE X
ASPARTATE AND IT'S LEADING INTO
THE MAGENTA HELIX.
THE GLUTAMINE IS COUPLING THAT
MAGENTA HELIX TO THIS YELLOW
STRUCTURE WHICH IS PRESENTING AN
ARGININE CALLED THE ARGININE
FINGER BORROWED FROM THE RAS
LITERATURE.
THE ARGININE FINGER IS POINTING
TO A SUBUNIT NOT SHOWN HERE, THE
ONE BEFORE THIS ONE IN THE
ASSEMBLY, AND THE ARGININE IS
DOING JUST WHAT THE ARGININE
FINGER OF A RAS GAP DOES.
IT'S POINTING RIGHT AT THE
PHOSPHATE CENTER THAT'S GOING TO
BE HYDROLYZED, AND IT HAS TO BE
POSITIONED CORRECTLY FOR THE
SUBUNIT BEFORE THIS ONE TO
HYDROLYZE ATP.
SO THIS STRUCTURE HERE IS
COUPLING THE ARGININE FINGER TO
THE DEAD BOX.
AND THE MAGENTA HELIX IS ALSO
TOUCHING DNA.
AND THE ORANGE HELIX IS TOUCHING
BOTH THE CLAMP AND THE DNA.
SO IT'S INTEGRATING EVERYTHING
AND THAT'S WHAT THE GLUTAMINE
118 IS DOING.
I COULD TELL YOU MORE ABOUT
GLUTAMINE 118 BUT IN THE
INTEREST OF PRESENTING NEW
INFORMATION THAT ISN'T YET STUFF
THAT WE'VE SOLIDIFIED AND
PUBLISHED, I'M GOING TO MOVE ON
BUT FIRST I'M JUST GOING TO SAY
THAT THE POWER OF THIS PLATFORM
IS SUCH THAT SUBU COULD ASK A
VERY SIMPLE QUESTION, WHAT WOULD
HAPPEN IF YOU MUTE TATED MUTATED GLUTAMIN E
118 AND -- HOW QUICKLY COULD YOU
GET FUNCTION BACK IN JUST ONE
MUTATION.
AND THE ANSWER IS, YOU COULD.
SO WHAT THIS DIAGRAM SHOWS YOU
IS THE RESULT OF A DEEP
MUTAGENESIS SCREEN, IN WHICH
GLUTAMINE 118 WAS FIXED TO
ASPARAGINE.
IT'S A SORT OF -- LEAST DAMAGES.
THE ASPARAGINE IS THE LEAST
DAMAGING.
WHAT THIS DIAGRAM SHOWS YOU ARE
THE RESULTS OF TWO EXPERIMENTS,
TWO SATURATION MUTAGENESIS
EXPERIMENTS CONDUCTED
INDEPENDENTLY.
SO YOU JUST HAVE FITNESS ON THE
X AND Y AXIS ON A LOG 10 SCALE
SO ZERO MEANS IT HAS THE FITNESS
OF THE ORIGINAL Q118N MUTANT.
NEGATIVE NUMBERS MEAN THE
ADDITIONAL MUTATIONS JUST KILL
THE CLAMP LOADER AND POSITIVE
NUMBER MEAN YOU ACTUALLY GOT
BACK ACTIVITY.
SO 3 WOULD MEAN YOU GOT A
THOUSAND FOLD TIMES MORE
ACTIVITY THAN THE Q118N, AND SO
IN IN FACT, WHAT'S SHOWN HERE IS
A SINGLE MUTATION, Q118 AND
GLYCINE 143N, THAT BRINGS BACK
ACTIVITY IN TWO INDEPENDENT
TRIALS.
GLYCINE 143N IS A RESIDUE THAT'S
VERY INTERESTING BECAUSE IT'S
NOT ACTUALLY INTERESTING IN THE
ORIGINAL SATURATION MUTAGENESIS
EXPERIMENT.
SO FIRST OF ALL, WHAT IT DOES,
THIS GLYCINE RES DUAL RESIDUE 143 IS
LOCATED CLOSE TO WHERE GLUTAMINE
118 IS IN THE STRUCTURES, IT'S
REPLACED BY ASPARE ASPARAGINE, WHAT THE
SYSTEM DOES IS RESPOND BY
PUTTING BACK AN ASPARAGINE WITH
HYDROGEN BONDING CAPACITY RIGHT
NEXT TO IT.
WHAT'S INTERESTING IS TO LOOK AT
WHERE THIS RESIDUE IS AND WHAT
IT'S DOING.
SO FIRST OF ALL, GLYCINE 143,
THIS IS A RESIDUE THAT WILL COME
BACK IN THINGS THAT I'M GOING TO
BE DISCUSSING.
GLYCINE 143 IS NOT AT A SITE OF
CONSERVATION.
OR AT A SITE OF MUTATIONAL
SENSITIVITY.
WHICH MAKES SENSE BECAUSE IF
YOU'VE DAMAGED THE SYSTEM BY
MAKING A MUTATION THAT CAUSES
LOSS OF FUNCTION, THE RESPONSE
WILL PROBABLY COME EASIER FROM A
SYSTEM WHERE THE SYSTEM IS
TOLERANT.
SO THAT'S ONE THING.
SO IT'S AT A MUTATIONALLY
TOLERANT SITE.
THE SECOND THING IS IT'S AN
IDIOSYNCRATIC SITE.
BY THAT, I MEAN THE T4 CLAMP
LOADER HAS GLYCINE THERE, BUT
THE POPULATION TENDS TO HAVE
LYSINE OR ARGININE.
AND THAT MAKES SENSE BECAUSE IF
YOU SEE WHERE THAT 143 POSITION
IS, IT'S RIGHT NEXT TO DNA.
SO YOU CAN START TO SEE, I
SHOWED YOU EARLIER ARGININE
THAT'S CRITICAL IN THE T4 CLAMP
LOADER WHICH IN THIS CLAMP
LOADER AT ANOTHER POSITION,
THERE ISN'T AN ARGININE OR
LYSINE, IT'S FOR SOME REASON
GLYCINE.
SO YOU CAN SEE HOW THE SYSTEM IS
IDIOSYNCRATIC IN RECOGNITION OF
DNA AND THAT'S WHERE IT GETS
BACK THE CAPACITY TO REPAIR A
FUNCTION.
SO WE WERE CURIOUS ABOUT THIS
CAPACITY TO REPAIR FUNCTION AND
I WANT TO NOW TURN OUR ATTENTION
TO THE SUBUNIT AND THE T4 CLAMP
LOADER WHICH WE CALL THE CLASP.
IT'S JUST A SUBUNIT ALSO FOUND
IN THE RFC COMPLEX IN
EUKARYOTES.
IF YOU LOOK AT THIS CRYSTAL
STRUCTURE, I WOULD SAY AS A
STRUCTURAL BIOLOGIST THAT IT'S
THE MOST IMPORTANT SUBUNIT IN
THE CLAMP LOADER.
EVEN THOUGH IT'S NOT AN ATPASE
SUBUNIT.
SO LOOK AT WHAT IT DOING.
IT'S ACTUALLY CONNECTING TO BOTH
SIDES OF THE CLAMP.
SO IT'S OPENED THE SLIDING CLAMP
THE STRUCTURE, THE SLIDING CLAMP
IN GREY IS OPEN AND IT'S
TOUCHING BOTH SIDES OF THE OPEN
SLIDING CLAMP.
IT'S TOUCHING THE PRIMARY
TEMPLATE JUNCTION AND IN FACT IT
SPECIFICALLY RECOGNIZES THE
PRIMARY TEMPLATE JUNCTION
WITHOUT GOING INTO DETAIL, IT'S
DOING SOMETHING THAT ALL
HELICASES DO, USING A
HYDROPHOBIC RESIDUE TO WEDGE IN
BETWEEN THE STRANDS OF DNA, SO
IT'S VERY, VERY IMPORTANT
STRUCTURALLY.
SO IT'S A DISAPPOINTMENT OR
SHOCK TO LOOK AT THE RESULTS OF
THE SATURATION MUTAGENESIS SHOWN
HERE AS A HEAT MAP AND SEE THAT
IN FACT IT'S NOT MUTATIONAL
LISTENSTIVE.
WHAT THIS HISTOGRAM DOES IS SHOW
YOU THE DISTRIBUTION OF FITNESS
EFFECTS FOR EVERY SINGLE POINT
MUTATION ON THIS LOG 10 SCALE
AND YOU'LL SEE BASICALLY MOST
MUTATIONS DON'T DO ANYTHING EVEN
THOUGH THE MOST DISTURBING ONES
PROBABLY NOT VERY DISTURBING.
SO THAT GOES COUNTER TO THE
POINT THAT THIS GREEN STRUCTURE
SO SO CENTRAL AND SO IMPORTANT
AND IT SAYS, WHOA, IS IT THAT WE
CAN'T READ WHAT THE STRUCTURE IS
TELLING US?
SO NOW WE WONDERED, WHAT WOULD
HAPPEN IF WE TOOK THE CLAMP
LOADER WHICH IS RUNNING UNDER
IDEAL CONDITIONS IN E. COLI WITH
LOTS OF LURIA BROTH AND
EVERYTHING IS HAPPY.
WHAT WOULD HAPPEN IF WE TIPPED
IT OFF BALANCE?
AND WHAT SUBU DID WAS TO MAKE A
CHIMERIC CLAMP LOADER.
SO THIS IS HERE IN PURPLE ARE
THE SUBUNITS OF THE WILD TYPE T4 CLAMP LOA DER.
ALL THESE PURPLE SUBUNITS ARE
ENCODED BY ONE GENE, AND SO
THERE ARE FOUR COPIES OF THAT
GENE SO THERE ARE FOUR AAA+
ATPASES GENERATED.
HERE ARE DIFFERENT CLAMP
LOADERS, THERE'S SOME EUKARYOTIC
ONES, SO BACTERIOPHAGE ONES SO
HE CREATED CHIMERIC CLAMP
LOADERS IN WHICH HE SWAMPED IN A
AAA+ MODULE FROM ANOTHER CLAMP
LOADER.
THIS IS AN EXAMPLE OF A CHIMERIC
CLAMP LOADER WITH A AAA+ THAT
COMES FROM ANOTHER CLAMP LOADER.
THE SO CALLED COLLAR DOMAINS ARE
FROM T4 AND THE GREEN CLASP
SUBUNIT IS FROM T4.
AND THE SLIDING CLAMP IS FROM
T4.
NOW, WHAT HAPPENS IS THAT IF YOU
SWAP IN EUKARYOTIC CLAMP LOADERS
THAT ARE QUITE
SEQUENCE-DIVERGENT, YOU GET
COMPLEES LOSS OF FUNCTION.
BY THAT, I MEAN SUBU CAN'T
RECOVER ANY PHAGE.
IF YOU CAN'T RECOVER ANY PHAGE
TO SEQUENCE, YOU DON'T KNOW
WHAT'S GOING ON.
BUT FOR THIS PARTICULAR PHAGE
CALLED RR2, WHEN HE SWAPS THAT
IN AT 64% IDENTICAL IN SEQUENCE
TO T4.
VERY SIMILAR.
WHEN HE SWAPS THAT IN, HE GETS A
4,000 FOLD REDUCTION WHICH SEEPS
LIKE A LOT BUT IT ACTUALLY
ENOUGH TO PURIFY PHAGE AND
ACTUALLY DOING A SECOND ROUND OF
COMPETITION, HE'S ACTUALLY ABLE
TO GROW UP THE WINNERS AND
SEQUENCE THEM.
WHEN HE SEQUENCES THEM, THE
STHINGS
THINGS THAT EMERGE -- HE'S NOW
GOING TO MUTATE THE GREEN
SUBUNIT.
IN OTHER WORDS THE CHIMERAS
REPLACE THE ATPASE -- THE SYSTEM
IS NOW REALLY HOBBLED BUT HE
MAKES MUTATIONS IN THE CLASP
SUBUNIT AND ASKS HOW QUICKLY
DOES THE SYSTEM RECOVER.
THE GREAT THING IS THAT IT
RECOVERS VERY QUICKLY.
BUT BEFORE I SHOW YOU HOW MUCH
IT RECOVERS, LET FOCUS ON THIS
DIAGRAM HERE, WHICH IS A SMALL
PART OF THE FITNESS MAP OF
SINGLE SITE SATURATION
MUTAGENESIS THAT IS ALL POSSIBLE
KNEW
MUTATIONS.
OF IT'S A SMALL PART OF THE
CLASP SUBUNIT.
WHAT I WANT TO SHOW YOU NOW IS
THE DEEP BLUE.
THIS IS EXTREMELY GRATIFYING
BECAUSE IF YOU GO AND ANALYZE
THE DEEP BLUE, YOU'LL SEE THAT
IT MAKES SENSE.
SO OUR STRUCTURES WERE, IN FACT,
TELLING US WHAT THE POINTS OF
SENSITIVITY ARE.
THESE TWO RESIDUES THAT ARE DEEP
BLUE HERE ARE EXACTLY WHERE THE
CLASP SUBUNIT TOUCHES THE
SLIDING CLAMP.
SO IN FACT, THE SENSITIVITY CAN
NOW BE UNDERSTOOD IN TERMS OF
THE STRUCTURAL DETAILS THAT WE
KNOW FROM THE CRYSTAL STRUCTURE.
BUT YOU CAN ALSO SEE THAT NOW
THE SYSTEM HAS RED IN SEVERAL
PLACES.
AND IF YOU LOOK AT THE
DISTRIBUTION OF FITNESS OVER THE
WHOLE GREEN SUBUNIT, IT GOES
FROM THIS VERY NARROW
ESSENTIALLY TOLERANT
DISTRIBUTION TO A DISTRIBUTION
THAT SHOWS SUBSTANTIAL LOSS OF
FUNCTION BUT ALSO GAIN OF
FUNCTION IN MANY PLACES.
YOU CAN NOW INTERPRET THE GAIN
OF FUNCTION AND I'M JUST GOING
TO SHOW YOU ONE EXAMPLE.
SO THIS IS NOW LOOKING AT THE
CLAMP LOADER AND IN GREEN IS
THIS CLASP SUBUNIT.
HERE'S DNA.
AND HEERP HERE ARE TWO RESIDUES.
GLUTAMINE 26 IS TOUCHING THE
PHOSPHATE BACKBONE WITH DNA IN
OUR CRYSTAL STRUCTURE.
23 DOESN'T TOUCH THE BACKBONE.
IF YOU REPLACE THIS BY ARNLG
KNEE ARGININE
OR LYSINE, YOU GET A TENFOLD
INCREASE IN ACTIVITY.
IF YOU -- IN THE BACKGROUND OF
THE CHIMERIC CLAMP LOADER.
IF YOU COMBINE THEM, SUBU GETS
100 FOLD INCREASE IN ACTIVITY.
SO WHAT WE LEARNED FROM THIS IS
THAT THERE'S AN UNTAPPED
CAPACITY FOR OPTIMIZATION BUILT
INTO THE SYSTEM.
SO THAT IT'S REALLY ABLE TO
RESPOND VERY QUICKLY, BY QUICKLY
I MEAN A SINGLE POINT MUTATION
STARTS WAKING YOU UP.
THE REASON THIS HAPPENS IF WE
THINK ABOUT IT IS OBVIOUS VEES
THAT ANY SYSTEM DRIFT AWAY
THROUGH NEUTRAL MUTATIONAL
DRIFT.
FROM THE HIGHLY OPTIMIZED SYSTEM
WHEN IT'S OPERATING AND IT'S
REDUNDANT IN ITS FUNCTIONS AND
YOU DON'T SEE THE CAPACITY FOR
RESTORATION OF FUNCTION UNLESS
YOU STRESS THE SYSTEM.
SO THAT'S WHAT WE WANT TO LEARN
FROM THIS.
NOW I WANT TO END BY MOVING BACK
TO THE DEAD BOX AND SHOWING YOU
WHAT WE LEARNED THAT WAS
INTERESTING AND SURPRISING FOR
US FROM THE STUDIES OF THE DEAD
BOX.
SO I'VE EXPLAINED WHAT THE DEAD
BOX DOES AND I WANT TO TURN TO
THIS LAST ASPARTATE IN THE DEAD
BOX WHICH I SAID COORDINATES AN
INTERFACIAL RESIDUE, IT'S
ARGININE 122 COMING FROM A
NEIGHBORING DOMAIN.
THE DEAD BOX AS I SAID IS FOUND
IN RNA HELI CASES, THAT'S WHERE
THEY WERE FIRST DEFINED, AND
FROM THE YEARS OF WORK DONE IN
RNA HELI CASES, WE KNOW WHAT
THIS ASPARTATE IS DOING IS
COUPLING RNA RECOGNITION TO ATP
HYDROLYSIS.
SO WE INFER THAT'S WHAT IT MUST
BE DOING IN THE CLAMP LOADER AS
WELL.
AND HERE IS A DIAGRAM TAKEN FROM
A PICTURE IN -- OR TAKEN FROM A
PAPER PUBLISHED IN NATURE ABOUT
10 YEARS AGO, AND IT'S
EXPLAINING HOW A DEAD BOX HEL
CASE WORKS, BUT THE WAY TO
REALLY THINK ABOUT THEM ARE
THEY'RE TWO ATPASES ON THE SAME
CHANGE SO LIKE TWO CLAMP LOADER
SUBUNITS.
IN THIS DIAGRAM FROM THE PAPER,
YOU CAN SEE THAT ATP IS BOUND
AND RNA IS BOUND AND ONLY WHEN
BOTH ARE BOUND IS AN ACTIVE SITE
CONFIGURED APPROPRIATELY WHICH
WILL TRIGGER HYDROLYSIS.
AN ION PAIR FORMED BETWEEN THE
LAST D OF THE DEAD BOX MOTIF AND
AN ARGININE PRESENTED BY THE
SAME HELICAL ASSEMBLY I'VE BEEN
FOCUSING ON IN THE CLAMP LOADER.
THIS IS WHAT THE LAST D OF THE
DEAD BOX IS DOING.
SO WE ASKED, WHAT WOULD HAPPEN
IF WE MUTATED THE D AND THEN
ASKED HOW -- OH, SOMEBODY CLOSED
THE CLOCK THAT WAS THERE THAT I
WAS KEEPING MY EYE ON.
PLEASE PUT THAT BACK BECAUSE --
HAVE YOU GIVEN ME 10 MORE
MINUTES OR WHAT DID YOU DO?
>> NO, I WAS TRYING TO ADJUST --
YOU GO AHEAD.
>> I THINK THE TIME IS ACCURATE.
YOU DON'T NEED TO ADJUST IT.
IT 3:38 BY MY WATCH AND THAT
CLOCK BUT IT'S EASIER TO WATCH
THE CLOCK SO THAT I CAN END AND
LET EVERYBODY GO.
WITHOUT RESENTMENT AND ANGER.
SO DON'T PUT THAT AWAY.
SO YOU KNOW, SO WE WANTED TO ASK
WHAT WOULD HAPPEN IF YOU MUTATE
THE LAST ASPAR TATE AND SEE HOW
QUICKLY THINGS COME BACK.
SO WHAT SOMEBODY TOLD ME IS THAT
YOU CANNOT WAKE THE DEAD, BUT
YOU CAN WAKE THE WALKING DEAD.
SO WHAT KENDRA MARCUS WHO IS
ASKING THIS QUESTION DID WAS TO
TRY TO IDENTIFY A MUTATION IN
THE ASPARTATE THAT WOULD BE THE
WALKING DEAD.
AND I'D ALREADY POINTED THIS OUT
TO YOU IF YOU CHANGE AS PART
TATE 110 -- IF YOU THINK ABOUT
IT A LITTLE BIT, YOU CAN SEE
THAT THE REASON THIS IS THE CASE
IS THAT THE ASPARTATE CAN -- THE
CYSTINE CAN DEPROAT NATE, YOU'LL
GET S MINUS AND THAT'S A MIMIC
FOR ASPARTATE.
IN FACT, KENDRA DID THE CRYSTAL
STRUCTURE OF THE ASPARTATE TO
CYSTINE MUTANT CLAMP LOADER
COMPLEX BOUND TO DNA, FROM
LOOKING AT HOW THAT CYSTINE
COORDINATES THE ARGININE, WE'RE
PRETTY SURE THAT'S WHAT'S
HAPPENING.
SO THIS IS THE WALKING DEAD.
IT'S TAKEN A HIT IN ACTIVITY BUT
WE CAN ASK HOW QUICKLY DOES IT
RECOVER.
SO NOW THAT MUTATION IS FIXED
AND KENDRA MUTATES EVERYTHING
ELSE USING THIS ASSAY.
SO FIRST JUST TO SHOW YOU IT
REALLY IS THE WALKING DEAD, WHAT
THIS GRAPH IS SHOWING US IS TIME
ON THE X AXIS AND ATP HYDROLYSIS
ON THE Y AXIS.
IF YOU ADD THE CLAMP LOADER AND
DNA TOGETHER, YOU GET NO ATP
HYDROLYSIS.
YOU NEED TO LOAD THE CLAMP
LOADER, DNA AND CLAMP, YOU GET
ATP HYDROLYSIS.
ATP HYDROLYSIS IS ONLY ONE SMALL
WINDOW ON THE FULL FUNCTION OF
THE WINDOW BUT IT'S A USEFUL
ONE.
IF YOU MAKE THE AS PART TATE 110
TO C MUTATION YOU'LL SEE IT'S
THE WALKING DEAD.
IT'S NOT DEAD, IT'S NOT
COMPLETELY ACTIVE AND IT'S
INTERMEDIATE, SO NOW WE WILL FIX
THAT MUTATION AND LOOK AT HOW
THE SYSTEM RESPONDS.
AND THE RESULT OF THIS WAS
ACTUALLY SO DISAPPOINTING, IT
HELD ALL OF US WORKING AND
THINKING ABOUT THIS BACK I WOULD
SAY A YEAR, WHICH IS SHOWN IN
THIS DIAGRAM, WHICH IS THAT
THESE RED SPHERES OR SURFACES
SHOW YOU THE SITES WHERE THERE
ARE SINGLE POINT MUTATIONS THAT
WAKE UP THE WALKING 10, MEANING
D110C THE THE MAJOR CONCLUSION
YOU CAN DRAW FROM LOOKING AT
THIS DIAGRAM IS THAT THESE SITES
OF RESCUE MUTATIONS ALL OCCUR IN
REGIONS THAT ARE TOLERANT TO
MUTATION IN THE ORIGINAL CLAMP
LOADER.
SO THAT'S NICE, THAT'S
GRATIFYING BUT IT'S NOT VERY
EXCITING.
AND WHAT'S PARTICULARLY
FRUSTRATING ABOUT THIS -- THESE
RESULTS IS THAT WE'VE SPENT
MONTHS STARING AT THEM AND WE
JUST CAN'T UNDERSTAND WHAT THESE
MUTATIONS ARE DOING.
THEY JUST MAKE NO SENSE TO ME.
SO ONE OUT HERE IS TO SAY, WELL,
THERE'S NO DIRECT RELATIONSHIP
BETWEEN STRUCTURE AND THE FREE
ENERGY LANDSCAPE ON WHICH THE
PROTEIN OPERATES, SO THESE ARE
JUST CHANGING THE FREE ENERGY
LANDSCAPE BUT I FOUND THAT
UNSATISFACTORY BECAUSE I KNOW
THAT'S TRUE, BUT I'D STILL LIKE
TO UNDERSTAND HOW THESE
MUTATIONS ARE AFFECTING THE
FUNCTION AND THEY REALLY DO --
THE ONES THAT KENDRA HAS
PURIFIED AND TESTED REALLY DO
AFFECT THE FUNCTION.
AS WE SHOW YOU HERE.
SO LET'S LOOK AT THIS ATPASE
ACTIVITY GRAPH FIRST.
SO AGAIN, THIS IS TIME VERSUS
ATP HYDROLYSIS AND THE WILD TYPE
IS IN BLUE -- IN BLACK AND WHEN
YOU ADD DNA AND THE CLAMP AND
ATP, IT HYDROLYZES ATP.
IN YELLOW IS D110C, WHICH I CALL
THE WALKING DEAD, WHICH HAS AN
ATTENUATED ATPASE ACTIVITY.
BUT IF YOU MAKE A SINGLE POINT
MUTATION, ONE OF THOSE RED
RESIDUES, YOU'VE WOKEN UP THE
WALKING DEAD.
THIS ATP ASSAY COMES BACK TO
WILD TYPE ATPASE ACTIVITY.
THIS GRAPH -- THE MUTATIONAL
FITNESS IS A LITTLE HARDER FOR
YOU TO TAKE IN BUT I'M JUST
GOING TO SHOW YOU A COUPLE
THINGS.
FIRST, IT'S SHOWING YOU THE
FITNESS OF VARIANCE, IN THE
BACKGROUND OF D110C, COMPLETELY
WILD TYPE CLAMP LOADER.
SO THIS IS PROLINE 50.
WHEN IT'S PROLINE, THAT MEANS
THE ONLY MUTATION IS D110C IS
LIGHT BLUE, THAT MEANS IT'S
TAKEN A LOSS OF FUNCTION.
THAT'S THE D110C FUNCTION, IN
OTHER WORDS.
WHY WOULD WHITE WOULD BE TOTAL WILD TYPE
PROTEIN WITHOUT THE D110C IS
ZERO OR WHITE IN THIS SCALE.
AND SO WHAT THAT MEANS IS THAT
IF YOU CHANGE PROLINE 50 TO
LYSINE, ACCORDING TO THIS
EXPERIMENT, YOU ACTUALLY -- NOT
ONLY DO YOU WAKE UP THE WALKING
DEAD, YOU ACTUALLY NOW -- THE
WALKING DEAD HAS WOKEN UP AND IS
RUNNING A MARATHON.
IT'S ACTUALLY A BETTER CLAMP
LOADER THAN THE FULLY WILD TYPE.
THIS IS THE ONE THING I'VE BEEN
ABLE TO RATIONALIZE A LITTLE
BIT.
THIS IS THE SITE OF PROLINE 50
IN THE SO CALLED FAMOUS P LOOP,
BECAUSE HERE'S THE PHOSPHATE,
THE PHOSPHATE BINDING LOOP.
HERE'S THE PROLINE.
THIS PROLINE WHICH IS ALSO A
SITE OF TOLERANT IDIOSYNCRATIC
SUBSTITUTION IS RIGHT NEXT TO A
GLUTAMATE.
SO YOU CAN SEE WHY MAKING IT A
LYSINE MIGHT GIVE YOU BACK
ACTIVITY BUT IT'S VERY
UNSATISFACTORY BECAUSE ARGININE
DOESN'T WORK.
BUT THE KEY TO UNDERSTANDING
WHAT ALL OF THESE MUTATIONS ARE
DOING IS TO LOOK AT THIS DIAGRAM
AND ACTUALLY APPRECIATE THAT
MANY, MANY, MANY SUBSTITUTIONS
ARE BETTER THAN THE ORIGINAL
WILD TYPE.
IN THE D110C BACKGROUND, PROLINE
TO LYSINE ISN'T THE ONLY
SUBSTITUTION THAT'S WAKING UP
THE WALKING DEAD.
YOU CAN ACQUIRE ACTIVITY TOWARDS
ORIGINAL WILD TYPE BY CHANGING
PROLINE TO MANY THINGS.
SO IT CAN'T JUST BE THIS ION
PAIR.
THIS WAS THE KEY THAT LET ME
GRIN
BEGIN TO UNDERSTAND WHAT THESE
RESCUE MISSIONS ARE DOING.
AND I UNDERSTOOD WHAT THEY'RE
DOING BECAUSE MOST OF MY LAB
WORKS ON SIGNALING PROTEINS LIKE
RAS AND EGF RECEPTOR.
WE STUDY MUTATIONS ALL THE TIME
BECAUSE THEY MANIFEST AS PATIENT
RESISTANCE MUTATIONS TO DRUGS.
BASICALLY THIS IS THE SIGNATURE
OF AN INHIBITORY INTERACTION
THAT'S BEING BROKEN BY THE
MUTATION, ALLOWING TO ESCAPE
FROM AUTO INHIBITION AND REGAIN
ACTIVITY THAT THE DAMAGE DONE BY
D110C HAD CAUSED TO INCUR.
THIS LETS US USE A PRINCIPLE WE
USE WHEN UNDERSTANDING KINASES
AND RAS AND THINGS LIKE THAT,
WHICH -- OH, I JUST WANTED TO
SHOW YOU, IF YOU LOOK AT THE
WHOLE HEAT MAP, JUST LOOK AT THE
LOWER GRAPH LIKE HERE.
YOU'LL SEE THAT MANY MUTATIONS
WAKE THE WALKING DEAD.
I DON'T WANT TO WALK THROUGH
THIS WHOLE COMPLICATED DIAGRAM.
THE PRINCIPLE WE USE TO
UNDERSTAND, I CALL THE
ANAKARANINA PRINCIPLE.
I READ A REVIEW BY LUIS JOHNSON,
INSPIRATION TO MANY OF US WHO
DIED SOME YEARS AGO BEFORE HER
TIME, AND IN HER OWE OBITUARY
PUBLISHED BY THE ROYAL SOCIETY,
THEY SAY -- THE WRITER SAYS SHE
WOULD BRING PROTEIN STRUCTURES
TO LIFE BY MAKING THEM POP LAT
CHARACTERS IN A NOVEL.
SHE'S QUOTING TO SAY HAPPY
FAMILIES ARE ALL ALIKE.
EVERY UNHAPPY FAMILY IS UNHAPPY
IN ITS OWN WAY.
AND THE PRINCIPLE HERE IS THAT
WHEN YOU HAVE AN ENZYME SYSTEM,
THE CATALYTIC CENTER HAS TO BE
CONSERVED IN THE HAPPY STATE,
WHICH IS THE ON STATE, BECAUSE
IT HAS TO OBEY THE DICTATES OF
CHEMISTRY.
BUT IF EVOLUTION WANTS TO
REGULATE THAT ENZYME, ALL THAT
EVOLUTION HAS TO DO TO CREATE AN
AUTO INHIBITED STATE THAT'S OFF
IS TO DO SOMETHING THAT MAKES
THE ENZYME UNHAPPY.
THE WAY EVOLUTION WILL DO THAT
FOR A DIVERGENT SET OF PROTEINS
WILL BE UNIQUE FOR EVERY
DIVERGENT PROTEIN OR EVERY
SUBCLASS.
AND WE SEE THAT IN KINASES,
THAT'S A LECTURE FOR ANOTHER
DAY.
SO WE STARTED THINKING ABOUT THE
ANNA KARENIN A PROCESS, WE'RE
MISSING A STRUCTURE.
THE STRUCTURE WE'RE MISSING IS
AN OFF-STATE OF THE CLAMP
LOADER.
AND TODAY, WE HAVE THE POWER OF
CRYO EM THAT WE CAN DEPLOY TO
ANSWER A QUESTION LIKE THIS.
WITH CRYSTALLOGRAPHY, WE COULD
BARELY OBTAIN THE CRYSTAL
STRUCTURES I'VE SHOWN YOU
BECAUSE -- I'VE ONLY SHOWN YOU
GLIMPSES OF BECAUSE THE CLAMP
LOADER IS SO DIFFICULT TO
CRYSTALLIZE.
WE DEFINITELY HAD NO VIEW OF THE
STRUCTURE FOR THE T4 SYSTEM AND
WE COULDN'T USED WHATEVER PEOPLE
HAD GUESSED ABOUT FOR OTHER
SYSTEMS TO UNDERSTAND THESE
MUTATIONS.
SO FORTUNATELY, THEY STARTED
APPLYING CRYO-EM TO DETERMINE
STRUCTURES OF UNCROSS LINKED
CLAMP LOADERS.
REALLY AS CLOSE TO THE NATIVE
STATE AS WE CAN.
THE FIRST RESULT SHOWN HERE IS
THE STRUCTURE OF THE HAPPY STATE
OF THE CLAMP LOADERS.
BOUND TO DNA, BOUND TO ATP
ANALOG, BOUND TO THE CLAMP.
AND IT LOOKS JUST LIKE THE
CRYSTAL STRUCTURE WE'VE
PUBLISHED MORE THAN 10 YEARS
AGO.
SO THAT'S A RELIEF FOR US, WE
DON'T HAVE TO REWRITE ANY OF OUR
CONCLUSIONS.
BUT HE WAS ALSO ABLE, VERY
QUICKLY, TO DETERMINE STRUCTURES
WHERE ONE OR THE OTHER THING IS
MISSING SO THE SYSTEM IS
UNHAPPY.
WHENEVER HE DOES THAT, HE GETS
THE SAME STRUCTURE BACK.
WHEN HE OMITS DNA, WHEN HE OMITS
THE CLAMP, WHEN HE USES ATP,
WHEN HE PUTS THE D110C MUTATION
IN, HE GETS THE STRUCTURE ON THE
RIGHT.
AND THIS IS AN UNHAPPY
STRUCTURE, IN FACT, IT'S A
STRUCTURE THAT'S VERY STABLE, A
DOESN'T NEED A CLAMP AND IT'S IN
A CONFIGURATION WHERE IT CAN'T
BIND DNA.
TO SHOW YOU THAT, HERE'S AN
ANIMATION INTERPOLATION BETWEEN
THE OFF OR UNHAPPY STATE AND THE
ACTIVE STATE.
IT'S ACTUALLY FASCINATING TO
LOOK AT THIS, YOU'LL SEE HELICES
UNWINDING AND THE REAL POINT I
WANTED TO MAKE FROM THIS IS THAT
THERE MUST BE AN ENERGY COST TO
GOING FROM THE INACTIVE STATE TO
THE ACTIVE STATE, THAT ENERGY
COST IS PAID BY DNA BUT ALSO BY
THE ARGININE ASPARTATE
INTERACTION SO PRESUMING WHAT'S
HAPPENING IS THESE MUTATIONS ARE
LOWERING THAT ENERGY COST.
WHAT'S REALLY EXCITING, IF WE GO
IN AND LOOK AT THE STRUCTURAL
CHANGES HAPPENING INSIDE ONE OF
THE ATPASES, OF COURSE THESE
GLOBAL CHANGES ARE ALSO
IMPORTANT, BUT IF WE LOOK AT ONE
OF THE STRUCTURAL CHANGES
HAPPENING INSIDE THE ATPASE, WE
CAN SEE THINGS WE CAN APPRECIATE
FROM THE STUDIES OF RAS WHICH
GUIDE SO MUCH THINKING IN MY
LAB.
SO TO SET THAT UP, HERE'S ONE
SUBUNIT OF THE CLAMP LOADER IN
THE ASSEMBLY, IN THE HAPPY
STATE.
HERE'S AN ATP ANALOG BACK HERE,
AND HERE IS THE TERMINAL
PHOSPHATE COLORED YELLOW AND
BLUE, HAPPENS TO BE -- FLUORIDE.
HERE'S A RESIDUE CALLED
ASPARAGINE 139 THAT'S ATTACKING
THE PHOSPHATE.
ASPARAGINE 139 IS EQUIVALENT TO
GLUTAMINE 61, THE SITE OF THE
THREE FAMOUS CANCER MUTATIONS IN
RAS.
WHAT ASPARAGINE 139 IS DOING IS
THE SAME, IT'S ON SWITCH 2, AND
IT'S DEPROAT NATEING A WATER
MOLECULE OR ACTIVATING A WATER
MOLECULE FOR ATTACKING THE
PHOSPHATE.
THIS IS A CONFIGURATION THAT'S
PERFECTLY POISED TO DO THAT.
WE'VE TRAPPED IT USING ATB
BERYLLIUM FLUORIDE.
YOU'LL SEE TWO ARGININE
RESIDUES, IT IS INTERACTING WITH
THE ATP IN THE SUBUNIT BEFORE,
HERE IT IS, AND THIS IS ARGININE
122, WHICH IS INTERACTING WITH
THE ASPARTATE OF THE DEAD BOX IN
THE PREVIOUS SUBUNIT.
SO IT'S ALL SET UP.
AND IF YOU GO THROUGH THE
SUBUNIT, THERE'S ANOTHER
ARGININE IN THE AAA PLUSES
CALLED SENSOR 2 AND IT'S
INTERACTING WITH THE PHOSPHATE
BUT NOW SETTING UP THE
INTERACTION WITH THE NEXT ATP.
SO THIS IS THE STRUCTURE WE
UNDERSTAND.
NOW IF YOU NO YOU LOOK AT AN
INTERPLAITION BETWEEN THE OFF
STATE DRIVED FROM THE
3.2 ANGSTROM CRY YOE EM
STRUCTURE, YOU START TO SEE TO
THOSE OF US WHO STUDY THESE
REALLY MARVELOUS CHANGES.
SO FOR EXAMPLE HERE, THAT'S THE
HAPPY STATE.
YOU SEE ASPARGINE 139 PULLED
AWAY BECAUSE IN THE OFF STATE,
THERE ARE NO FLANKING
INTERACTIONS.
THEY'RE ALL BROKEN IN THE OFF
STATE.
SO THIS IS REALLY OFF BECAUSE
THE CATALYTIC RESIDUE HAS BEEN
PULLED OUT.
AND THE OTHER THINGS YOU COULD
LOOK AT, HERE'S GLUTAMINE 118
RESIDUE, I SPENT A LOT OF TIME
DISCUSSING EARLIER, BUT SUDDENLY
WHAT YOU SEE IS THAT THESE
RESCUE MUTATIONS, THE MOST
IMPORTANT ONES SHOWN AT RED
SPHERE, ARE IN REGIONS WHERE THE
STRUCTURE IS CHANGING.
SO I DON'T KNOW WHAT THEY'RE
DOING THROUGH THE INNER JE TICS, THE ENERG ETICS,
BUT I SEE THEY'RE LOCATED IN
SITES WHERE THE STRUCTURE
CHANGES BETWEEN OFF AND ON.
SO ALL THEY HAVE TO DO IS ALTER
THAT ENERGETIC BALANCE OR THE
LANDSCAPE OR THE BARRIER, AND WE
CAN UNDERSTAND WHY THEY CAUSE
THE WALKING DEAD TO WAKE UP.
SO WITH THAT, I'LL END WHAT
WE'VE LEARNED FROM OUR STUDIES
IS THAT THERE IS, OF COURSE, RES
RESIDENT IN EACH OF THESE
PROTEINS ENORMOUS CAPACITY FOR
SEQUENCE CHANGE.
WHILE THE PHAGE IS REPLICATING
DNA.
THAT'S REALLY THE MOST IMPORTANT
FUNCTION.
HYDROGEN BONDED STRUCTURAL
ELEMENTS MAINTAIN COUPLING
ACROSS INTERFACES AND AND I INLT I
INTRODUCED THE ANNA CARE NINA
PRINCIPLE, IT'S DOING SO
DIFFERENTLY BECAUSE THE OFF
STATES CAN BE DIFFERENT.
I THINK THE STRUCTURES CHANGE
AND AS THEY CHANGE, THEY'RE
MOVING ALONG DIFFERENT
EVOLUTIONARY TRAJECTORIES.
IT'S PART OF WHAT HAS ENABLED
MANY THINGS -- THE DIVERSITY OF
THESE STRUCTURES, WHICH I'VE
SHOWN YOU THREE HERE, CLAMP
LOADER, CLPX UNFOLDASE USING THE
SAME ELEMENTS TO CONTROL THEIR
ACTION.
WITH THAT I'LL END, AND ONCE
AGAIN THANK SUBU SUBRAMANIAN,
KENDRA MARCUS, KENT GORDAY,
YOUNG JAN JUAN AND CHRISTINE GEE
FOR BEING INSTRUMENTAL IN
LETTING THIS WORK TAKE PLACE AND
BE DONE.
SO THANK YOU.
[APPLAUSE]
>> THANK YOU FOR THE GREAT TALK.
I WAS CURIOUS, IN YOUR RESCUE
EXPERIMENT WITH THE D110C, DO
YOU EVER HAVE DOUBLE MUTATIONS
FOR EXAMPLE THAT RESCUE MORE
EFFICIENTLY THAN THE SINGLE
MUTATIONS?
>> SO THE QUESTION IS, WITH THE
D110C, HAVE WE STUDIED DOUBLE
MUTATIONS.
AND SO THE SIMPLE ANSWER IS NO,
BECAUSE THE WAY THE EXPERIMENT
WAS DONE WAS TO ONLY INTRODUCE
ONE MUTATION AT A TIME.
SO -- I ALLUDED TO THE FACT IN
THE RESCUE OF THE SO-CALLED
CLASP SUBUNITS, SUBU DID FIND HE
COULD COMBINE TWO OF THE
MUTATIONS GIVEN ADDITIONAL.
SO AS YOU CAN IMAGINE, THE
DIVERSITY OF POSSIBLE SEQUENCES
EXPLODES WHEN YOU START
COMBINING MUTATIONS, SO ONE OF
THE THAINGS WE'RE PROBABLY GOING
TO DO IS NOT SIT AND COMBINE
THESE MUTATIONS BUT USE VARIOUS
METHODS FOR GENERATING
ALTERNATIVE SEQUENCES THAT ARE
VASTLY -- NOT COMBINING JUST TWO
MUTATIONS, BUT MIXING UP LOTS OF
THINGS AND THEN -- BECAUSE THIS
IS A HIGH-THROUGHPUT EXPERIMENT,
WE CAN TEST THOUSANDS OF
VARIANTS IN AN EXPERIMENT, AND
FROM THAT GLEAN SOMETHING ABOUT
COUPLING BETWEEN RESIDUES.
>> THANK YOU.
>> JOHN, THANKS FOR A PHENOMENAL
TALK.
SO I WAS WONDERING ABOUT SORT OF
TWO THINGS.
ONE IS, I'M THINKING ABOUT THE
WORK THAT HAS BEEN DONE IN TERMS
OF THE STATISTICAL COUPLING
ANALYSIS --
>> SORRY, I JUST WANT TO
INTERRUPT BECAUSE YOU MENTIONED
HIM AND I SHOULD REALLY HAVE PUT
HIM ON THE SLIDE BECAUSE HE'S
RESPONSIBLE FOR TRANSFERRING --
HE'S THE PERSON WHO TAUGHT MY
LAB HOW TO DO THESE EXPERIMENTS
AND SUBU SUBRAMANIAN IS THE
UPPER LEFT-HAND CORNER HERE WAS
A GRADUATE STUDENT WITH RAMARAN.
SO WE REALLY OWE RAMA FOR HIS
INTELLECTUAL LEADERSHIP IN
GETTING US TO THIS POINT.
>> RIGHT.
SO I IMAGINE THAT THE -- IN THE
FIRST PART, THE CONSERVE CLUSTER
YOU SHOW THAT WOULD PROBABLY
COME OUT OF A STATISTICAL
COUPLING ANALYSIS.
>> YES.
>> I'M CURIOUS, THE LAST PATCH
OF THE SCATTERED RESIDUE THAT
YOU SHOWED, WOULD THEY ALSO SHOW
UP IN AN --
>> THEY WOULD NOT.
AND YOUR QUESTION IS A GREAT ONE
BECAUSE IT LETS ME FURTHER
EXPAND WHAT I THINK IS
CONCEPTUALLY THE IMPORTANCE OF
WHAT I CALL THE ANNA KARENINA
PRINCIPLE.
SO ALL HAPPY FAMILIES HAVE TO BE
HAPPY IN THE SAME WAY.
AND SO WHEN YOU DO A SEQUENCE
CONSERVATION OF A LARGE FAMILY,
THE SEQUENCE CONSERVATION IS
GOING TO REFLECT THE HAPPINESS
STATE.
SO YOU SEE THE BLUE BUILT WHICH
IS REALLY COUPLING CATALYSIS.
NOW YOU USE THE ANNA KARENINA
PRINCIPLE AND YOU LEARN THAT
EACH UNHAPPY FAMILY IS UNHAPPY
IN ITS OWN WAY.
I REFER TO THAT AS I HAD YOE AS I HAD YOE SIN AS IDIOSYNCRASY.
IF YOU HAVE A LARGE DATABASE,
SOME FAMILIES WILL BE HAPPY IN
THE SAME WAY.
FROM OUR WORK ON SIGNALING, OUR
WORK ON SIGNALING IS REALLY WHAT
LED US TO JUMP TO THIS
CONCLUSION, IS TO CONSIDER
THE -- AND I'M JUST GOING TO SAY
THIS BECAUSE SIGNALING IS REALLY
WHAT MOST OF THE LAB DOES, THE
KINASES THAT HAVE SH2, SH3 AND
KINASE ARE ANCIENT FAMILY OF
SIGNALING KINASES.
BUT THE SUBMEMBERS OF THIS, THE
BTK KINASE HAVE REALLY DIFFERENT
WAYS IN WHICH THEY USE THE
SH2 AND SH3 DOMAINS TO SWITCH
OFF THE KINASE.
SO THEY'RE UNHAPPY IN DIFFERENT
WAYS.
OF COURSE IF YOU ENTER THE SARC
KINASE LINEAGE YOU'LL FIND WHAT
MAKES THEM --
>> I HAVE A SECOND QUESTION.
THIS IS ABOUT THE GLYCINE THAT
BASICALLY GETS MUTATED TO SORT
OF THEN RESCUE THE FIRST
MUTATION, IN THE FIRST PART OF
YOUR TALK.
SO MY QUESTION IS THAT IF YOU
CONSTRAIN THAT -- IF YOU NOW DO
AN EXPERIMENT WHERE YOU DON'T --
THAT YOU DON'T LET THE GLYCINE
MAKE A CHANGE INTO THE -- I
THINK IT WAS CHANGING INTO A
GLUTAMINE, HAVE YOU DONE
EXPERIMENTS LIKE THAT AND NOW --
SO ESSENTIALLY IT'S -- TO WHAT
EXTENT CAN IT ACTUALLY RECOVER,
IF YOU START CONSTRAINING TWO
RESIDUES?
I DON'T KNOW IF I MAKE MY
QUESTION CLEAR.
>> WE HAVEN'T DONE THE
EXPERIMENT QUITE THE WAY YOU'RE
SAYING, BUT WE KNOW FIRST FROM
JUST THE NATURAL SEQUENCE
VARIATION IS THAT THERE ARE
PROTEINS THAT DON'T USE THAT
GLUTAMINE AND DON'T -- SO NATURE
IS VERY DIVERSE IN COMING UP
WITH SOLUTIONS.
AND THE SECOND THING I'D SAY IS
THAT WE CAN, OF COURSE, THINK AS
I SAID EARLIER OF COMBINING THIS
WITH THAT WITH THIS BUT WE
HAVEN'T QUITE GOTTEN GOING WITH
THOSE.
>> THANK YOU.
Q.JUST
>> JUST BECAUSE THE COMBINATIONS
ARE SO LARGE.
>> OKAY.
SO FROM ONLINE, TOM SCHNEIDER
ASKS, HOW MUCH ATP IS USED PER
STEP OR REMOVAL, QUESTION MARK,
OF THE CLAMP LOADER?
>> YES.
I'M ASKED HOW MUCH ATP IS USED
IN THE CYCLE OF WORK, NOT OUR
WORK BUT MIKE O'DONNELL HAS
SUGGESTED IN PAPERS -- THAT'S A
DIFFICULT QUESTION TO ANSWER
BECAUSE WHAT YOU END UP DOING IS
MAKING MUTATIONS AND SO YOU
CAN -- THE ANSWER IS THAT YOU
CAN GET A FUNCTIONING CLAMP
LOADER IF ONLY ONE ATP IS
HYDROLYZED.
SORT OF HOBBLING ALONG.
>> ANY MORE QUESTIONS?
ONE MORE COMING DOWN?
>> THERE'S SOMEBODY COMING DOWN
HERE OR THERE.
NO, PLEASE.
>> ABOUT THE GLYCINE MUTATION,
YOU KNOW IT'S NOT ONLY THE --
BUT IT'S ALSO THE VIBRATIONS AND
THE MOTION OF THIS, THE GLYCINE
DIFFERENTLY IS BASICALLY
ALLOWING TO BE MORE FLEXIBLE
THAN THAT.
>> YES, OF COURSE THE GLYCINE
INTRODUCES FLEXIBILITY WHICH IS
REMOVED BY THE MUTATION THAT
RESCUES AND THE DYNAMICS
CANNOT --
>> ALSO THE -- IS GLYCOSYLATED.
>> THE T4 DNA IS GLU COST LATED
AND THAT'S ONE OF THE THINGS
THAT THE PHAGE MACHINERY DOES
AFTER IT INFECTS THE PHAGE AND
ACTUALLY THAT'S THE REASON WE
HAD TO PUT IN A CRISPR SYSTEM,
BECAUSE THAT PREVENTS US -- YOU
SEE THE PHAGE INFECTS THE
E. COLI, THEN I DIDN'T GET ASKED
THE QUESTION WHY DIDN'T WE JUST
SEQUENCE THE E. COLI THAT'S LEFT
OR SOMETHING LIKE THAT.
BUT THE THING IS THE PHAGE COMES
IN AND SO DAMAGES THE DNA, THE
HOST DNA.
AND THAT'S THE REASON WE PUT IN
THE CRISPR SYSTEM TO GET THE
PHAGE OUT.
SO FULLY APPRECIATE THAT.
YEAH.
>> A QUESTION ABOUT THE FIRST
PART OF THE TALK.
WHEN YOU DID THE FIRST
MUTATIONAL ANALYSIS, SOME OF THE
MUTANTS ACTUALLY ENHANCE THE
PROPERTIES, THE REPLICATION OF
THE T4, SO THAT MEANS THAT
NATURE, IF EVOLUTION, YOU KNOW,
WENT ON LONGER, COULD ACTUALLY
FIND MORE EFFICIENT T4s THAT
COULD --
>> YES, NATURE COULD FIND MORE
EFFICIENT T4 WHICH IS SURELY THE
CASE BECAUSE WE'RE SO QUICKLY
ABLE TO CHANGE THE FUNCTION
AFTER A MUTATION.
SO THAT LEADS INTO DIFFICULT
QUESTIONS OF WHAT IS ACTUALLY
THE CONDITION AL SPECTRUM UNDER
WHICH THE PHAGE IS ACTUALLY
BEING SELECTED.
>> AND ONE OF THEM WAS THE
PHENYL RESIDUE INTO DEXD MOTIF
TO --
>> LIKE WHY WOULD THAT WORK
BETTER THAN WILD TYPE.
SO WE DON'T KNOW.
WE SHOULD MAKE THAT.
BUT MAYBE I'M TRYING TO
COMMUNICATE THE SENSE THAT THESE
DATA ARE RICH AND CAN BE
EXPLOITED IN MANY WAYS.
>> ABSOLUTELY.
THANK YOU.
>> THANK YOU.
>> ALL RIGHT.
I THINK WITH THAT, LET'S THANK
JOHN FOR A REALLY, REALLY
INTRIGUING AND ENGAGING TALK.
[APPLAUSE]
>> THANK YOU ALL.