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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5319_Библиотеки_им_академика_М_И_Перельмана.pdf
Chapter • Three-Dimensional Structure of Biomolecules
14
. Fig. 14.6 The polymer chain of aglobular protein reverses its di-
rection in the loop or turn area. Numerous turn patterns have been
found. They are made up of 2–6 amino acids. Normal turns (left) form
aC═O⋯HN hydrogen bond (violet) in the direction of the polymer
Proteins organize their secondary structural segments
into motifs. For example, the sequence of an α-helix,
aβ-strand, and another α-helix forms amotif. Multiple
motifs fold into domains to yield the tertiary structure
of aprotein. Domains may be preferentially made up of
helices, folded sheets, or acombination of both building
blocks. Often the domain has aspecic function. Many
chain. This hydrogen bond has adifferent order in inverse turns (cen-
ter). Another group of open turns (right) is held together by van der
Waals contacts and polar interactions
proteins consist of asingle domain. Complex proteins
can be made up of multiple domains. When acomplex
assembly of several separate polymer chains is formed
(e.g., as in hemoglobin), it is called aquaternary structure.
Despite the enormous multiplicity that can be achieved
by combining the 20proteinogenic amino acids into sequences, there seems to be arather limited number of folding possibilities for the domains. How many total folding
patterns exist can only be speculated. Of all the crystal
structures known today, about 1200 different folding patterns have been found. This number is mainly based on
. Fig. 14.7 The course of the polypeptide chains is symbolized with
spirals for α-helices, with arrows for β-pleated sheets, and with threads
for different turn segments. Approximately 30% of the structural-
ly known proteins can be assigned to one of the nine shown folding
classes. The rst folding pattern (upper left) is a“TIM barrel,” and the
second one is an “open pleated-sheet” structure
a
. • From Secondary Structure Via Motifs and Domains to Tertiary and Quaternary Structure
b
c
. Fig. 14.8 The folding pattern of different β-barrel structures can
be thought of as apolymer chain with eight separate β-strands (ar-
rows). These are separated by loop areas. Upper row An up-and-down
barrel forms when the folding of the polymer chain of eight β-strands
follows a zigzag pattern. The antiparallel sections form hydrogen
bonds between themselves that close up to form a cylinder. Middle
row The four-β-strand polypeptide chains lie next to one another so
that the rst chain interacts with the fourth, and the second interacts
data from globular enzymes and transport proteins. Since
hardly any new examples have been found in recent years
despite intensive efforts, it can be assumed that there are
perhaps 1500–2000 stable folding patterns used by Nature.
Considering these numbers, it must be taken into account
that there are many intrinsically disordered proteins that
with the third. Then the double strand folds and the rst pair comes
to lie next to the second. Because the course of the polymer chain
is reminiscent of the engravings on Greek vases, the pattern is called
aGreek key. Two such patterns can come together into acylinder-like
orientation and form aGreek-key barrel. Bottom row Another folding
pattern is formed from adouble strand that is placed together with
an internal twist. The double strand wraps itself into acylinder-like
structure that is called ajelly roll
can adopt an ordered fold only when they are fullling
their function. Very little is known about their structural
organization. It remains to be seen whether new folding
patterns can be found here. Using their computer programs, David Baker's group (University of Seattle, USA)
was able to design proteins with novel folds and to predict
ab
Chapter • Three-Dimensional Structure of Biomolecules
the sequence that folds into that spatial structure. As a
proof-of-concept, the group then synthesized these proteins and, as predicted, their designed architecture was
different from all previously known proteins in Nature.
This opens up the possibility of creating an enormous variety of new “articial” proteins that can be used as drugs,
vaccines, enzymes or sensors. For his achievements, David
Baker was awarded the Nobel Prize in Chemistry in 2024.
About 30% of all folded proteins belong to one of the
classes shown in . Fig.14.7. From the group of membrane-bound proteins, perhaps 1000 structures are known so
far, although about 30% of all human proteins fall into this
group. Based on such limited data, it seems difcult to make
reliable predictions about possible additional folding classes.
Drug design focuses on the interaction of aligand with
aprotein. As aresult, the structural considerations of
chemists are usually limited to the amino acid groups that
protrude into the binding pocket. However, the folding
pattern around the binding pocket inuences the properties found there. For example, ahelix oriented towards the
binding pocket determines the local electrostatic potential.
This can be exploited to design selective ligands that bind
only to proteins of aparticular folding class.
Despite the progress in structure determination techniques, it can happen that the structure analysis of an
important protein fails, but the structure of arelated protein, for example, can be solved. On this basis, amodel of
the desired protein can be built (Sect.20.5). This requires
information about the structure and folding principles
of proteins. They allow us to understand which part of
the protein stabilizes the scaffold, which parts determine
the function, and which parts account for the differences
between homologues.
Adetailed discussion of these principles would go too
far here. As an example, consider the folding pattern of the
β
-barrel. An extended sheet of multiple β-strands has an
internal twist (see . Fig.14.5). For example, if eight such
strands are lined up side by side, acylinder can be formed.
This barrel-like folding pattern of eight or more strands is
often observed. Several variations of this folding pattern
are shown in . Fig.14.8, which illustrate how and accord-
ing to which principles apolypeptide can fold spatially.
A loop acts as a connecting element between the
pleated sheet strands of the β-barrel in the example in
. Fig.14.8. α-Helices can also serve as connecting elements
(. Fig.14.7). In the center abarrel-like structure forms
14
. Fig. 14.9 The amino acid groups that determine the folding pat-
tern and those that carry the function are located in different regions
of the protein. Left The catalytic site (yellow spheres), which binds and
transforms substrates lies in aTIM-barrel-type structure (
red cylinder, β-strands: light-blue arrows) at the end of the barrel where
one would expect to nd alid. The loops of the polymer chain that
surround this “lid” (gray and green threads) carry the function-deter-
mining amino acids. Right The function-determining amino acids in
α-helices:
the loop region occur in the open-pleated-sheet structure there, where
the attached helices change from the top to the bottom of the pleated
7 https://sn.pub/5hAz72)
sheet. (
ab
. • Are the Fold Structure and Biological Function of Proteins Correlated?
. Fig. 14.10 Immunoglobulins form ahighly specic binding pocket
in which they recognize antigens. These are exogenous substances. The
enormous structural diversity of these binding pockets is achieved by
variations in the amino acids in the loop regions. Left The immunoglobulins have aY-like shape, which is divided into atrunk (constant
Fc domain) and two identical Fab branches. Right The course of the
polymer chain in these branches corresponds to the barrel type. The
antigen binding site is found on the right-hand side. The displayed
polypeptide chain covers the region circled in the schematic drawing
from β-strands, and on its outer surface the bridging α-helices are aligned. This folding pattern was rst discovered
in triosephosphate isomerase. It is therefore called aTIM
barrel (. Fig.14.7). Another important folding class that
is made up of α-helical and β-pleated sheet segments com-
prise the open-sheet structures (. Fig.14.7). In this class,
the pleated sheet does not close to acylinder but rather it
remains open. Helices group above and below the sheet.
How does the structure of aprotein relate to its function?
For example, do all proteases have the same folding pattern? Alarge number of enzymes with distinctly different
functions all belong to the TIM barrel type or open-sheet
structure. There are many oxidases, isomerases, kinases,
aldolases, synthases, dehydrogenases, or proteases that can
be assigned to these two classes. Here, Nature started from
acommon origin and developed divergently. Consequently,
the function of aprotein is not necessarily coupled to apar-
ticular folding pattern. When the structure of the enzyme
is further analyzed, it is found that the catalytic sites of the
proteins of afolding class are located at the same position.
This is found at the C-terminal end of the barrel in the
TIM barrel structure and at the topological switch of the
connecting helices from the top to the bottom of the open
on the left. At the right end (colored) are the loops responsible for
recognition of exogenous substances. They grasp the antigen (dark red
molecule) like the ngers of two hands. (7 https://sn.pub/Soz3AF)
sheet structure (. Fig.14.9). The function-determining
amino acids are located in the loop region between adjacent folded sheets and helices. Why would Nature follow
this principle of separating folding structure from function? The amino acids that enable the stable folding of
adomain are separated from those that induce aspecic
function. This is avery efcient evolutionary strategy. Two
regions have been optimized simultaneously:
The stability of the protein scaffold in special folding
-
patterns and
The layout of the amino acid sequence to serve aspe-
-
cial function.
By spatially separating and displacing the functional
groups in the structurally less committed loop regions,
the two tasks could be optimized in parallel. Changing
asingle amino acid in asecondary structural element
could destabilize the entire folding pattern and stop folding. This will be avoided if the amino acid sequence to be
functionally optimized is placed on astable scaffold that
does not interfere with the optimization.
Aclass of proteins that implements this principle to
perfection are immunoglobulins. As antibodies, they recognize and bind to foreign substances called antigens. To
remove an antigen, immunoglobulins with highly specic
binding pockets and high afnity must be available within
afew days. The substances recognized can be anything
from small organic molecules to large proteins. To achieve
this goal, it is estimated that approximately 1012 different
14
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0
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0
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0
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0
3
Chapter • Three-Dimensional Structure of Biomolecules
. Fig. 14.11 The side chains of apeptide substrate and the binding
pockets that they belong to are classied on the N-terminal side of the
peptide as P3, P2, P1… or S3, S2, S1… (left); on the C-terminal side they
are classied as
,
,
… or
,
,
… (right)
antigen sequences are produced. Considering that each
antibody-producing cell makes just one specic immunoglobulin and that the human genome contains only
about 105 genes, how can this difcult task of achieving
such diversity be accomplished? It is solved by cells of the
immune system using acombination of different variable
gene segments and excessive amino acid exchange in these
segments during lymphocyte maturation. In this way,
variable loop regions are formed on astable scaffold of
barrel-shaped pleated sheet structures (. Fig.14.10). The
therapeutic value of such biomolecules (called biologicals) has been recognized. Many monoclonal antibodies
are nowadays available as therapeutics (Sect.32.3).
Proteases cleave polypeptide chains during enzymatic degradation or release an active protein or peptide from an
inactive precursor form. For this purpose, the enzymes possess acatalytic site at which cleavage takes place (Sect.14.6
and Chaps.23,24,and25). In order to specically recognize
aparticular substrate, several binding pockets are located
on their surface. These are structurally complementary to
the side chains of the substrate, which are oriented around
the catalytic sites. In 1967, Israel Schechter and Arieh
Berger proposed anomenclature to describe these pockets (. Fig.14.11). The positions of the amino acids of the
peptide substrate are described as P3, P2, P1–
,
,
, and
so on. Starting from the N-terminus, position P1 is immediately before and position
is immediately after the cleavage
site. The binding pocket of the enzyme for the side chain
of amino acid P1 is called S1, and the same is valid for the
other side chains. This very useful nomenclature is purely
formal. Applying these labels to aparticular enzyme does
not mean that all of the pocket actually exists. Two binding
pockets can appear as one large binding pocket in the 3D
structure. The S3 and S4 binding pockets in the serine protease thrombin are actually just one large pocket (Sect.23.3).
It is also possible that asubstrate amino acid does not have
acomplementary binding pocket in the enzyme. It then protrudes into the surrounding water environment.
Peptides are easily synthesized with enormous diversity
(Sect. 11.5). If the peptide is attached to a probe that
changes its color or uorescence upon release (Sect.7.2),
the labeled peptide can be used to determine the substrate
prole of the protease. To do this, alarge library (Sect.11.1)
of these peptides is exposed to the protease, and the members that are well cleaved are identied. . Fig.14.12 shows
the amino acid composition of alabeled tetrapeptide that
is preferentially cleaved by the proteases trypsin, factorXa,
plasmin, and chymotrypsin. Peptides with basic groups
such as arginine or lysine are preferentially cleaved by trypsin, plasmin, and factorXa. Factor Xa almost exclusively
converts peptides with arginine at the P1 position. Chymotrypsin behaves quite differently. It prefers aromatic amino
acids such as tyrosine, phenylalanine, and tryptophan at
the P1 position. The selectivity at positions P2 to P4 is not
nearly as pronounced. Trypsin will convert tetrapeptides
with branched groups at P2 such as Phe, Tyr, Trp, Ile, or Val
much more poorly if an arginine is at the P1 position. Basic
groups are also less favored. Trypsin shows virtually no selectivity at P3 and P4. Factor Xa has aspecial preference for
the small glycine at position P2, but there is hardly any difference for the groups at position P3 for this enzyme. On the
other hand, different groups are more strongly selected at
the P4 position. The substrate binding prole helps to reveal
the selectivity characteristics of enzymes. They show the
complementary properties in the binding pocket and help
to inspire initial ideas for the design of possible inhibitors.
This concept was applied to cysteine proteases in the
research group of Jonathan Ellman at the University of
California at Berkley, USA. Substrate molecules were synthesized that carried auorescent marker at the end of an
amide bond to be cleaved. Various organic building blocks
were attached to the other side. When such asubstrate molecule is cleaved by the protease, the organic moiety must be
bound in the binding pocket of the enzyme. Therefore, the
transformation indicates the binding of atest molecule. The
method is ideal for screening purposes. Ahit discovered in
this way can easily be chemically converted from asubstrate
molecule to an inhibitor. For example, by replacing the
cleaved amide bond with an aldehyde function, acysteine
protease inhibitor (Sect.23.9, . Fig.23.27) that has very
little in common with the peptide substrate can be designed.
. • From Substrate to Inhibitor: Screening of Substrate Libraries
a
b
. Fig. 14.12 Upper rows A tetrapeptide library, held constant in
position P2 to P4, was varied at position1 (red) with 19 amino acids (one-letter notation; nnorleucine). It is cleaved by trypsin after
arginine and lysine, by factorXa after arginine, and by plasmin after lysine. Chymotrypsin prefers aromatic amino acids at position P1.
Bottom rows If arginine is held in position P1 and the remaining three
positions (blue, green, orange) are varied, trypsin shows practically no
selectivity for the amino acids at P2, P3, and P4. On the other hand,
factorXa prefers aglycine in position P
2
ab
Chapter • Three-Dimensional Structure of Biomolecules
What information about the dynamics and reactivity
of molecules can be extracted from acrystal structure?
Molecular vibrations are visible even in the solid state.
This is reected in the blurriness of the electron density.
When amolecule participates in areaction, bonds are
broken and new bonds are formed. The formation and
cleavage of amide bonds is acentral task in biochemical
processes. Molecule 14.2 contains an amide and an ester
group (. Fig.14.13). When acrystal of this compound
is exposed to thermal energy, asolid state reaction occurs
to form 14.3. The molecule is in ageometry in the initial
crystal structure that is conducive for entering into the
reaction pathway.
. Fig. 14.13 If thermal
energy is applied to acrystal
of 14.2, the carbonyl group of
the ester function reacts with
the amide NH2 group and an
imide bond is formed between
N1 and C8 to give 14.3. There
must be an implicit vibrational motion that culminates
in the reaction. At the same
time, the ester bond between
C(8) and O(2) is cleaved
during these reaction steps
Information about changes in the geometric orientation of functional groups in the chemical reaction is critical to understanding the concomitant structural changes
that occur. This knowledge is aprerequisite for the design of transition state analogue inhibitors (Sects. 6.6
and22.3). With respect to the formation or cleavage of
an amide bond, the question is: from which direction
does the amino group attack the carbonyl carbon in the
course of nucleophilic addition to form anew bond?
In the early 1970s, Hans-Beat Bürgi and Jack Dunitz
at ETH Zurich in Switzerland began to extract information about the geometric changes along such reaction steps
from crystal structures. Before the days of movies and
television, people came up with creative ways to make
pictures move, such as ip-books (. Fig.14.14). They
give the impression that adynamic story is being told.
Imagine that the pages of the book have fallen apart
14
. Fig. 14.14 A story is shown in
static pictures in ip-books. If the different pages of this story ip pass the
eyes quickly enough, the impression
of adynamic process will be given
. • Solutions to the Same Problem: Serine Proteases with Diering Folds Have Identical Function
sign (Chap.20). In addition, this information is needed to
compare the binding properties of molecules (Chap.17)
or to explore binding pockets for their preferred ligand
binding sites (hot spot analysis). The Isostar database,
assembled and maintained at the Cambridge Crystallographic Data Centre in England, contains many such
contact geometries and spatial distributions.
. Fig. 14.15 The formation or cleavage of an amide bond occurs by
nucleophilic addition. Anucleophile, for instance, an oxygen or nitrogen atom, approaches the planar carbonyl carbon atom. During the
reaction it rises out of the plane of the three neighbors and adopts
atetrahedral conguration. Examples were sought from low molecular weight crystal structures in which anitrogen atom approaches
acarbonyl group between asingle bond and avan der Waals contact
in the crystal packing. By superimposing these data, it is recognizable
that the approach of the nucleophilic nitrogen towards the carbonyl
group is “perfomed from back and behind.” Simultaneous with this
spatial approach, the carbon atom migrates out of the plane towards
the nucleophile. The geometric requirements of this reaction step also
determine the structural composition of the catalytic center of avariety of hydrolases (Sect.22.3)
from frequent use and they are now in disorder. You
need to put them back in order. In this case, ordering
criteria are needed. Asimilar task is posed by organizing
structural data to describe areaction. From databases of
known crystal structures (Sect.13.9), particular crystal
structures are sought in which an amino group is near
acarbonyl group, as in the structure of 14.2. Finally, they
are put into alogical order (. Fig.14.15).
The systematic comparison of crystal structure data
provides a preliminary information about structural
molecular properties, for instance, about the preferred
conformation (Sect.16.4). The geometry of noncovalent
interactions can also be evaluated in this way. The side
chain of the amino acid histidine contains an imidazole
ring with its two nitrogen atoms. In the neutral state, one
of these nitrogen atoms is ahydrogen-bond acceptor and
the other is ahydrogen-bond donor. There are hundreds
of molecules with an imidazole ring in the database of
low molecular weight crystal structures. In these structures, the imidazole ring actually has acceptor and donor
interactions, usually with neighboring molecules. All of
these structures are superimposed on one another based
on their common imidazole ring (. Fig.14.16). It shows
in which spatial direction the hydrogen-bonding partner
of the imidazole nitrogen atoms are located. The task
of estimating the possible interaction positions in the
binding site of the protein for the functional groups of
aligand is undertaken in the course of denovo drug de-
It was shown in Sect.14.4 that the amino acids that determine the folding and function of aprotein occur in
separate parts of the structure. However, for enzymes
with the same function, Nature has found the same solution by using different spatial foldings.
The function and therapeutic signicance of serine
proteases will be discussed in more detail in Chap.23.
Aset of three amino acids, the so-called catalytic triad,
plays akey role in accelerating the hydrolysis of amide
bonds by these enzymes. The two amino acids serine
and histidine and one acidic amino acid residue, such as
aspartic or glutamic acid, are found in acharacteristic
spatial orientation. They are dened by the narrow limits
imposed by the reaction geometry required for nucleophilic addition (Sects.14.7 and23.2). Their composition
is ideal for the cleavage of amide bonds.
. Fig. 14.16 The crystal packing of low molecular weight com-
pounds affords an overview of possible interaction geometries of hydrogen-bond donors (left) and acceptors (right) around the nitrogen
atoms of an imidazole ring. Accordingly, all structures with an imidazole ring were sought in which at least one of the two nitrogen atoms
participates in ahydrogen bond. The superposition of the structures
shows where the positions of the interacting partners can be expected.
(7 https://sn.pub/G6cGKS)
Chapter • Three-Dimensional Structure of Biomolecules
14
. Fig. 14.17 Trypsin(a, red) and subtilisin(b, green) are serine prote-
ases. They have the same catalytic triad of serine, histidine, and aspartic acid. These function-determining amino acids are, however, placed
upon entirely different folding patterns In(c), the course of the chain
of both proteins is superimposed upon one another. Despite this, the
side chains of the amino acids of the catalytic triad are in the same
spatial position(d). The course of the polymer chains are shown with
The enzyme trypsin is composed of two barrel-like
subunits (. Fig.14.17a). The catalytic site is located
at the interface between these two subunits. Subtilisin
is another serine protease that belongs to the class of
open-pleated sheet structures. The catalytic triad occurs in aloop region at the edge of the folded sheet
(. Fig.14.17b). If the amino acids involved in catalysis
are removed from the protein and superimposed in space,
the identical geometry of the triad becomes apparent. In
addition to the enzymes mentioned, this catalytic triad
is also found in lipases and esterases (Sect.23.7), which
colored ribbons that represent the spatial orientation of side chains of
the three catalytic amino acids. (7 https://sn.pub/9m77Ot)
also cleave peptide or ester bonds. Although they show
divergent scaffold folding, the geometric orientation of
their triads is once again identical.
Our genetic information is encoded in the DNA molecule. It is athread-like molecule about 20 Å in diameter
and up to 2 m long when stretched. It has the shape of
adouble helix (. Fig.14.18). On the outside, apolymer
. • DNA as aTarget Structure of Drugs
chain of sugar and phosphate building blocks wraps
around the base pairs like abanister. The latter bases
form acomplementary pair at each step. The base pairs
are linked together by aspecic hydrogen-bonding pattern. Apurine base (adenineA and guanineG) always
interacts with apyrimidine base (cytosineC and thymineT; in the related RNA molecule, thymine is replaced
by uracilU; . Figs.14.18 and14.19). The resulting spiral
staircase has apitch of 34 Å and reaches afull turn after
ten steps. The two intertwined polymer strands form two
grooves of different sizes on their surfaces (. Fig.14.18).
When the DNA is examined from the side along the steps
at the major and minor grooves, the characteristics of
the base pairs become visible. In the minor groove, there
are three functionalities that determine the interactions
with other molecules. In the major groove there are four
such functionalities. Interestingly, the pattern that is read
in the major groove is unambiguous because of the exposed properties for each base pair on astep. In the minor groove, only the overall difference between AT/TA
or GC/CG can be distinguished (. Fig.14.19), but not
their spatial sequential topology.
The base pairs on three neighboring steps code for
one amino acid each (see Sect.32.7). In order to read this
information unambiguously from the DNA, proteins
that regulate gene expression (so-called transcription
factors) read the information from the major groove,
from the side (see Sect.28.2). Only there is it possible to
read the prescribed code (AT, TA, GC, CG) unambiguously. The DNA molecule is highly charged due to the
many outward-facing phosphate groups. This charge is
neutralized by the formation of ion pairs, mostly with
. Fig. 14.18 The DNA molecule is made up of individual stair steps.
One pair of bases forms each step. The sugar phosphate chain suspends the steps like adouble banister. It forms amajor and aminor
groove on the surface. Left A segment of DNA with 14 base pairs.
Center A schematic representation with the sugar–phosphate backbone as agray arrow, guanine (light green), cytosine (violet), thymine
(cyan), and adenine (red). Right Amodel of aDNA surface in which
the size difference between the minor and major grooves is empha-
sized. The individual bases align according to their interaction properties (blue H-bond donor, red H-bond acceptor, gray hydrophobic
contact). (7 https://sn.pub/LGZoaa)