........
.
.
.
.
.
.
.
_
REFLECTION
TtBS 22 - OCTOBER 1 9 9 7
shown to react with a chMromethy~ketone enzyme inhibitor~.The N-terminus
of the B chain (]le16), created by the
activation cleavage, was found buried in
company with an aspartate (Azp194) in
an unprecedented internal salt bridge.
did not foresee the tremendeus pressure that Nature would exert to get a
rapid publication. Had I been older and
was once exclusively ours, and those wiser, I would have ignored it, to proceed
are memories we shall cherish in old in my own time. But with sterling help
age. It is achievement on the journey; from our 'computing assistants', a group
not flag-waving after finishing, that has of young lad~es who had worked with us
for years measuring densRometer traces
always thrilled me.
and punching IBM cards (Fig. 3), and
from the laboratory artist Annette Snazle
Paul S[gler, Richard Henderson and I (now Lenton), we got a paper together
set out energetically to interpret the map, describing the structure in general terms
using the amino-acid sequence already and Nature published it very quicPdy7.
determined by Brian Hartley~.4. (Michael
Rossmann, who took part in the early Activation mechanism understood,
stages ~, had left a couple of years be- August :~9~7
Many people imagine the structure of
fore, and Brian Matthews, who was pivotal, particularly on the computing side, chymotrypsin was then 'solved', but in
had left at the end of 1966.) A three- reality this was only the first act in a
dimensional model was built from brass long drama. The low pH side of chymo'Kendrew' components, and detailed the trypsin's activity range could be underchain trace that was engraved deeply into stood as titration of His57, but why did
my mind (Fig. 2). This map clearly showed the activity fall so rapidly above pH 8.5?
His57 adjacent to the active site Ser195, A possible answer was mentioned in our
giving a comforting agreement with ex- 1967 paper, but there were puzzling inpectation, because this histidine had been consistencies with the published data.
The tortuous story o[ Asp°..fIiso.°Sef:
structural analysis of x-chymotfypsin
One Monday evening in March 1967, more
than six years after i had started work on
tha crystal st~uctme o~ (~-chymoh°ypsin,
we began to plot an electron-density
map using our novel computer-controlled
high-precision cathode ray tube ~ at the
Laboratory of Molecular Biology in
Cambridge, UK. We had spent the Sunday
night at the iBM 7090 computer at
Imperial College, London, OK, merging
our data and calculating the electron density. The average of the electron densities for the two independent molecules
in the crystal asymmetric unit was calculated, giving a map of improved accuracy. The resulting sections of contour
map were recorded on 35 mm film over
Monday night, and transparencies were
printed on Tuesday (Fig. 1). [ had a
chance for a quick look at the map in the
late Miernoon, and decided to come back
after dinner to spend more time with it.
! was late home that night. We already
knew where the active site was, but 1
found ! could follow the peptide chain
from there through the whole molecule, ...... ( 5 :
0
::
with only small uncertainties. There was
a break at the autolytic cleavage site,
just where it should be expected, and
the disulphide bridges gave the highest
densities in the map, linking peptide
chains with the expected topology ~-.I arrived home thinking '1 know the structure of chymotrypsin. ! am the only person in the world who does'. Lying in
bed, 1 found ! could follow the whole
length of the chain in my mind.
! read a piece in a scientific journal a
few months ago which made me sad. The
writer described the onset of old age as
the gradual realisation that there would
• " " .i.
.. }- .: :, ,,.. " -_ :.:. :, .!/ ,,
:.:. ". ,-..2:
be no m,~re recognition for one's research
achievements. This appears to me so
wrong[ We do research because there is
unexplored territory out there, and the
journey is as irresistible as it was to
Francis Drake or Edmund Hiilary. From
time to time, every scientist gets to a
place no-one has ever been before. It is
thrilling to be there, it may be a whole
continent or a tiny speck, but every reFigure 1
search worker has had some knowledge
A section of the electron density map dated 20/3/67, as recorded on 3.6 mm film. This
section passes through the active site, and shows some of the density for Asp102
that has been theirs alone, until they
(then believed to be ASh), I-iis57, Tosyl-Ser195, Asp194 and (~-NH3+-Ile16. (Kindly provided
published it to the world. Even if the
by Richard Henderson.)
world ignores it, we never forget that it
reserved.
0968- ~004/97/$17.00 Pll:S0968-0004(97)01115-8
405
Copyright© 1997,ElsevierScienceLtd.Allrights
TIBS 22 - OCTOBER 1997
the positively charged sidechain of a trypsin substrate),
and there was no such substitution near the tosyl group.
But on the opposite side of
the active site, chymotrypsin's Asnl02 was substituted
by Aspl02 in the trypsin sequence. Residue 102 did not
look accessible from the active site, suggesting a puzzling conformational change
on substrate binding 9.
Map improvementand
interpretation,1988
Another [actor was that
the map did not s ~.em to be
of a high enough quality for
the resolution. For almost a
year, | worried about this
problem and found a sign
error in a program to refine
Figure2
heavy-atom positions, which
'Ribbon' diagram showing the main-chain structure of
could
have caused a heavy
oechymotrypsin. The chain termini shown at residues
atom
to
become misplaced, l
16, 146 and 150 are the result of activation and autoalso found that the refinement
lysis, which create three separate peptide chains in
procedure that we had been
the molecule, designated A, B and C. Disulphide
bridges are shown as shaded bars. The positions of
using converged too slowly.
the catalytically important residues 57, 102 and 195
After modified procedures ~°,
are highlighted in black. Apart from the N-terminal acwhich displaced one hafttivation peptlde and the C-terminal helix, the molecule
occupied heavy-atom site by
is composed of two topologically similar domains of
almost 2 A, a new map could
six 13-strands, represented by residues 23-124 and
be calculated. Although indi125-229. (Drawn by Annette Snazle.)
vidual map sections looked
almost indistinguishable, their
In July, ! went to a Gordon Conference, tiny differences caused the three-dimenand afterwards to visit several interested sional map to be amazingly improved, in
laboratories in North America, taking particular, extra density at the carbonyl
with me a ia~e pile of papers to read on groups of many peptide bonds allowed
aircraft and in hotel rooms, it was evi. the main chain conformation to be interdent that some of the data or interpre- preted more accurately.
tations were misleading, and the conSigler had left by now and, with my
clusion, blindingly obvious as it now new colleague Jens Birktoft, I set about
appears, took shape gradually in my building an accurate model from the
mind. Returning to England, ! found that new map. By now the Richards Box (or
Paul Sigler had independently reached Fred's Folly) had been invented n. Instead
the same opinion. The ile16...Asp194 of looking at a contour map and building
salt bridge was needed for activity, and a representation of it alongside, we could
the or-amino group began to be titrated use a half-silvered mirror to see the elecabove pH 8.5. The structural change for tron density superposed on the model as
activation was almost identical to the it was built. This was difficult because
change when chymotrypsin is brought one looked at the mirror image of what
to neutral pH from pH 9 (Re[. 8).
one was actually building, but performance improved with practice.
Pmles aboutthe bindingsite, 1968
After several months of careful model
,~md how did the substrate bind? Per- adjustment, Birktoft began to work syshaps there was a clue from a toluene- tematically through the molecule, recsulphonyl (tosyl) inhibitor bound to the ording the interactions made by each
active serine. But this clue appeared to sidechain, and considering the effects of
point in the wrong direction. We were sure amino-acid substitutions in chymotrypthat the difference between trypsin and sin B, trypsin and elastase, the other
chymotrypsin had to be an extra nega- serine proteinases whose sequences
tive charge at the PI binding site (to bind were then available to us ~z-~4.
406
GirktofL's reve{at[on and HartUey's
confirmation, eavay:L960
One day Blrktoft came to me and said
there was something puzzling that l
should come and look at. He showed me
chymotrypsin's Ash 102, whose ~-oxygen
was neatly hydrogen-bonded to the
~l-nitrogen of the active-site histldine,
His57. The problem is, he said, that although trypsin is highly homologous in
this part of its sequence, it has aspartic
acid at this point. The implications o{ that
buried aspartate did not take long to
consider. The histidine would be highly
polarised, and in its uncharged state
there would always be a proton on the
~l-nitrogen, never on the ~2-nitrogen,
which faces the active sedne, making
the latter strongly nucleophilic.
Within a few minutes, I was talking
to Brian Hartley, who had determined
the chymotrypsin sequence five years
earlier in Hans Neurath's laboratory a.
He said the sequence 100-102 (Ash-AshAsh) had given serious interpretation
problems with deamidation, and agreed
to redetermine this bit of sequence as
soon as possible. That redetermination
was done amazingly quickly, and confirmed our guesses. Although residues I00
and 101 are both asparaglne, residue
102 of chymotrypsin is aspartic acid.
For the first time, an enzyme structure
had revealed a new chemical mechanism. The catalytic machinery was plain
before our eyes.
Now there was another scramble to
submit a paper to Nature, this time because of the competition. We knew that
Joe Kraut's group had just completed a
structure for subtilisin, and he was coming over to present it at a Discussion
Meeting of the Royal Society within a few
weeks. We wanted to submit our new
discoveries before Kraut showed us his
results. In this paper, we presented a
range of possible interpretations of the
polarisations in what we called the
charge relay system. It was clear in any
case that an attack of Ser195 on the substrate peptide bond had to result in
transfer of a proton. A figure presented
the extreme possibilities, one being the
case where another proton is als,~ transferred from N~1(57) to O~(102). Although
we did not think this would happen, the
possibility was presented. We allowed
ourselves to forget that many readers
(almost all, you might think) look at figures carefully but skim the caption, never
reading the corresponding text. This figure led to the fallacy, perpetuated by
some textbooks, that our interpretation
depended on this proton transfer.
TIBS 22 - OCTOBER 1997
REFLECTI0P4S
Correction of a small error
in another figure (Fig. 4) meant
that publication was s~ight%,
delayed. Our paper ~'~, which
was to have bccn published
back-to-back with i@aut's paper
on subtilisi# 6, was actually
published a week ~ater.
Subst~ate binding, 1968-1060
This discovery removed the
supposed difference between
trypsin and chymotrypsin at
residue 102, and exploded the
theory that the PI sidechain of
the substrate would bind there.
We came back to the hypothesis that the tosyl group occupied the specificity site.
Tom Steitz had now joined
the group, and both he and
Henderson were searching for
conditions to bind substrate
into crystals. Henderson collected full high-resolution difRgure 3
fraction data on crystals of the
Planned originally as a record of the bulk of the 30-odd cartons of 10 000 IBM cards that comprised
'native' enzyme unsubstituted
the chymoptrypsin data, this photograph shows Paul Sigier, Brian Matthews and David Blow (in front),
with (left to right) the 'computers' Jill Collard (now Dawes), Diana Singleton (now Watson), Sue
by the tosyl group, soaked in
Simpson and Sue Wickham. There was only room for a few of the IBM cards (lower right). On the left is
formyl4.-phenylalanine. A difpart of the Joyce Loebl densitometer, which was used to measure every line ot diffraction spots on
ference electron-density map
several hundred X-ray precession photographs.
against the native showed no
density at the tosyl site, where
we expected to see phenylalanine. Equally, of dioxan, Henderson and SteRz tried re- plete set of high-resolution chymoSteitz, using a highly unreliable 4-circle moving dioxan from the supernatant of trypsin data on film had now been rediffractometer to study differences at low pregrown native crystals. Even at low res- duced from a person-year or so to about
resolution, failed to find sufficient differ- olution, these crystals showed significant two months. Steitz and Henderson measences when indole was substituted into differences, indicating that something had ured 2.5 A data from dioxan-free crys'native' crystals. Difference map after been removed from the tosyl site. The tals, which, together with earlier data
difference map from different compounds original 'native' crystals had
showed either no detectable binding or contained di~xan bound in
binding in the 'wrong' places - to only the specificity pocket, which
one of the two identical molecules at was displaced when indole
positions clearly created by the crystal was bound, largely masking
Cys58
~
Cys42
packing or in positions that explained the density for indole in differnothing of the well-studied specificity. ence maps. Low-resolution
Surprisingly, the inhibitor p-iodophenyi- difference maps against the
acetate clearly showed its iodine bound new data obtained from
dioxan-free crystals were
in the tosyl site.
Earlier studies of chymotrypsin had much more satisfactory, inbeen dogged by crystal twinning, in which cluding one for formyi4.-trypevery crystal grows with two different ori- tophan, a 'virtual substrate'.
entations of the crystal lattice, making the The dioxan had produced a
recording of diffraction data inaccurate great step forward for
Ser195
and unreliable, if not impossible. Barbara crystallisation but no-one
His57
Jeffery (now Harris) had discovered that had guessed it wouid bind at
0 Carbon
'~
twinning could be eliminated by crystal- the active site, obscuring
Q Nitrogen
lisation in the presence of 2% dioxan 5, the results of the substrate0 Oxygen
which was then routinely used in all binding studies.
0 Sulphur
Asp102
Using
the
computercrystallisation procedures before transhigh-precision
ferring the crystals into other solutions. controlled,
Rgure 4
Helped by a visit from Brian Matthews, cathode ray tube as a microBall and stick model of Asp...His...Ser as presented
who thought he remembered that the densitometer =~,and after the
in 1969. Figure originally drawn by Annette Snazie
original native data had been collected necessary development work,
and reproduced with permission from Ref. 15.
(five years previously) in the presence the time to measure a corn-
4O7
TIBS 22 -
collected from crystals substituted with
formyl¢-tryptophan and formyl-L-phenylalanine, produced new difference maps.
These maps revealed accurate density
for the pseudo-substrates in the pocket
occupied by the tosyl group in our original structure ~8.
sp~L, ey of~p=., Im 1~9
How, then, could trypsin be specific
for lysine or arginine at PI? Looking at
the pocket occupied by the tosyl group,
there was a significant sequence difference at the end of it. In the existing
trypsin
sequence 13'19, Ser189 of
chymotrypsin was replaced by 'Asn'189.
We guessed, and again Hartley quickly
confirmed, that trypsin really has aspartic acid at this point2°. Interpretation of
the original sequence data had falsely
indicated a deamidated asparagine.
Conclusion
Even this tortuous drama was only
the first in a series of exciting discoveries about the serine proteinases. The
long-running saga continues, as is documented in other papers in this issue.
The charge relay system (or catalytic
triad as some unaccountably describe
it) is a familiar feature of active sites,
and a histldine polarised by a buried
acid group even more common.
Some of the lessons I learned were:
(1) Be sceptical about published interpretations of data (particularly other
people's).
(2) Be particularly careful when under
pressure for rapid publication.
(3) Make sure your figures tell the
right story, even to someone who hasn't
time to read the caption.
Looking back, 1 savour the recollection of research leading to discovery,
puzzlement yielding to revelation, confirmation that apparently inexplicable
phenomena all fit into the scientific
scheme. Most of all, perhaps, I appreciate the satisfaction of sharing intense
effort and rewarding insight with colleagues who also became friends.
Acknowledgements
I thank several of my former colleagues, who have helped to refine my
recollections of what happened about
30 years ago.
OCTOBER 1997
5 Blow, D. M., Rossmann, M. G. and Jeffery, B. A.
(1964) J. Mot. Bio!. 8, 65-78
60ng, E. B., Shaw, E. and Schellrnan, G. (1965)
J. Biol. Chem. 240, 694-698
7 Matthews, B. W., Sigler, P. B., Henderson, R.
and Blow, D. M. (1967) t';ature 214, 652-656
8 Sigler, P. B., Blow, D. M., Matthews, B. W. and
Henderson, R. (3.968) J. Mol. Biol. 35,
3.43-.164
9 Blow, D. M. (3.969) Biochem. J. 3.3.2, 263.-268
./.0 Blow, D. M. and Matthews, B. W. (3.973) Acta
Crystallog, r. A29, 56--62
1"/Richards, F. M. (:1.968) .t. MoL Biol. 37,
225-230
-12 Smillie, B. et al. (3.968) Nature 23.8, 343-346
-13 Walsh, K. A., Kauffman, D. L.,
Kumar, K. S. V. 8. and Neurath, H. (.1964) Prec.
Natl. Acad. ScL Wash. 51,303.-308
.14 Shotton, D. M. and Hartley, B. S. (1970) Nature
225, 802-806
.15 Blow, O. M., Birktoft, J. J. and Hartley, B. S.
(3.969) Nature 221, 337=340
J.6 Wright, C. S., AMen, R. A. and F,raut, J. {1969)
Nature 221, 235-242
17 Arndt, U. W., Crowther R. A. and MaHett, J. F. W.
{1968) ./. ScL Inst. 3., 510-516
.18 Steitz, T. A., Henderson, R. and Blow, D. M.
(3.969) J. MoL Biol. 46, 337-348
29 Mikes, O. et at. (3.966) BioDhys. Biochem. Res.
Commun. 24, 346-352
20 Hartley, B. S. {:L970) Philos. Trans. R. Soc.
London Set. B 257, 77-87
References
2 Gossling, T. H. (3.967) Acta Crystallogr. 22,
465-468
2 Brown, J. R. and Hartley, B. S. ('1966)
Biochem. J. '103., 2'14-228
3 Hartley, B. S. ('1964) Nature 201, '1284-1287
4 Hartley, B. S. and Kauffmann, O. L. (1966)
Biochem. J. '105., 229-23'1
DAVaD ~. BLOW
81ackett Laboratory,Imperial College of
Science, Technologyand Medicine,
London, UK SW7 2BZ
Emaia:[email protected]
i TECHNICAL TIPS ONUNE
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£dt~r Adrian Bird, Institutefor Cell and MolecularBiologyat the Universityof Edinburgh