From the
In this paper we describe the results of molecular modeling of
the structures of the active sites of two subtilisin-like prohormone
convertases (SPCs), SPC2 (PC2) and SPC3 (PC1/PC3). These enzymes are
members of a recently discovered family of cellular proteases involved
in the processing of precursor proteins. Although these proteases all
possess catalytic domains similar to the bacterial subtilisins no
tertiary structural data from x-ray analysis are yet available. We have
shown that despite the high structural homology of the subtilisins and
the SPCs, the structure of the loop which lies immediately below the
active sites differs due to the presence of a cis-peptide bond
(Tyr
In neuroendocrine cells many peptide hormones and neuropeptides
are synthesized as part of larger, less active or inactive precursors.
An essential step in the formation of bioactive peptides is the
selective endoproteolytic cleavage of their
precursors
(1, 2, 3, 4, 5) , as
was first demonstrated in the processing of proinsulin to
insulin
(6) . Within the last few years a novel family of
cellular endoproteinases involved in precursor processing has been
discovered which includes the yeast mating pheromone processing enzyme
kexin
(7, 8) , the mammalian endoproteinases furin
(SPC1)
Surveys of the cleavage sites of a large
variety of precursors that are processed within the secretory pathway
have led to the conclusion that most of them possess complex multibasic
cleavage sites of the general type Arg-X-Lys/Arg-Arg
(R-X-K/R-R)
(17, 18) . Most prohormones on the
other hand are cleaved at paired basic residue sites, primarily Lys-Arg
or Arg-Arg. However, further analysis of sites cleaved in mouse AtT-20
cells, which contain SPC3 as their major endogenous protease, has
revealed that, in addition to the foregoing sites, others consisting of
Arg-X-X-Arg (R-X-X-R) are also cleaved
(19) .
Therefore, a basic residue at position -4 relative to the
scissible bond (P4) may play a very important role in enhancing
substrate site selectivity of the SPCs generally, as is illustrated
especially in the processing carried out by furin
(20) . Thus,
both furin and SPC3, and possibly also SPC2, catalyze the processing of
protein and peptide precursors containing Arg-X-Lys/Arg-Arg or
Arg-X-X-Arg cleavage sites in the constitutive and/or
regulated exocytic pathways.
When kexin was cloned and
sequenced
(7) , it was found to contain a catalytic domain that
is homologous to that of the bacterial subtilisins. Amino acid sequence
alignments of SPC2, SPC3, furin, and kexin showed a very high degree of
similarity in their catalytic domains
(4, 21) . The most
significant difference between the subtilisins and the catalytic
domains of the SPCs is the large increase in the number of negatively
charged residues (Glu and Asp), relative to the subtilisins, which may
contribute to the great selectivity of this family of enzymes for
substrates containing paired basic
residues
(5, 22, 23) .
Although x-ray
investigations of SPCs have not yet been carried out, molecular
modeling of the catalytic domain of furin has indicated that it is
closely related in its three-dimensional structure to the
subtilisins
(21, 22, 23, 24) . This
circumstance opens the way for molecular modeling and protein
engineering of the SPCs on the basis of their homology with
subtilisins.
The three-dimensional structures of four subtilases have
been established, subtilisin BPN`
(Novo)
(25, 26, 27) , subtilisin Carlsberg (27,
28), thermitase
(29) , and proteinase K
(30) . The overall
conformation of these enzymes is very similar
(21) , especially
in their active site regions
(27) . In molecular modeling the
active sites of SPC2 and SPC3, we have used the x-ray data on
subtilisins BPN` and Carlsberg
(27) . The substrate-binding site
of the subtilisins was identified by analysis of their crystalline
complexes with inhibitors or inactive analogs of
substrates
(26, 27) . The active site grooves of the
subtilisins can bind at least 6 amino acid residues
(P4-P2`)
Residues forming the active sites of subtilisin BPN` and
Carlsberg are indicated in . This information is taken from
Ref. 27, describing contacts of less than 4 Å between subtilisins
and the peptide inhibitors eglin-c or chymotrypsin inhibitor 2. The
presumptive residues at structurally equivalent positions in the
catalytic domains of SPC3 and SPC2 are also given in in
accordance with the amino acid sequence alignment of known subtilases
as proposed by Siezen et al.(21) . In accord with our
modeling results, we have included 3 additional amino acid residues
because their side chains potentially interact with residues P1 and P4
of substrates: 165 in subsite S1, and 131 and 135 in subsite S4 of SPC3
and SPC2.
However, we have noted a significant difference between
the primary structures of the subtilisins and PCs that appears to be
important in the structural organization of these proteins. In
subtilases with known x-ray structures, a cis-peptide bond
always occurs between residues 167-168 and in all cases residue
168 is proline, as is usually the case following this kind of
bond
(32) . Moreover, in dipeptide fragments of the type Tyr-Pro
(Tyr
In contrast to the
subtilisins, all the SPCs contain Ala or Thr instead of Pro at
positions corresponding to 168 of the
subtilisins
(21, 24) . This suggests that a
trans-, rather than cis-, peptide bond occurs in this
position. In most proteins cis-peptide bonds occur
exceptionally rarely preceding residues other than Pro
(33) ,
e.g. the loop formed by the sequence, Thr-Gln-Ser-Pro-Ser-Ser
(residues 5-10), of the Fab domain of immunoglobulin MCPC 603
contains a cis-peptide bond between Ser and Pro, whereas in
the analogous loop, Thr-Gln-Thr-Thr-Ser-Ser, of Ig F19 all bonds are
trans (files 1MCP and 1F19 of the Brookhaven Protein Data
Base). Therefore, the alignment of amino acid residues for the loop
165-171 given by Siezen et al.(21) in the SPCs
may be incorrect. Moreover, in their alignment the small Gly or Ala
residues at position 169 in the subtilisins are replaced by Asp (in
SPC3, ) or Asn (in kexin and furin), which is unrealistic
because the methyl group of the side chain of Ala
To appropriately align the
amino acid sequences of the subtilisins and SPCs, we have taken into
account the characteristic interactions of the side chains of the
residues Tyr
In
order to preserve the Tyr
To conserve structural correspondence between the two
loops,(165-171) and 320-327, in the preliminary stage of
molecular modeling we introduced range constraints on the arrangements
of the main chains of these two peptide strands between the C
Any theoretical study of enzyme-substrate
interactions should begin with considerations of factors which govern
the primary specificity. It is most practical to undertake this
initially at the stage of the formation of covalent complexes between
the catalytic side chains of the enzyme and the atoms of the scissible
bond of the substrate. Investigation of nonbonded Michaelis complexes
would make this problem significantly more complex since the
introduction of six additional variables for the orientation of the
nonbonded substrate as a whole relative to the enzyme would be
necessary.
Subtilisin, like other serine proteases, cleaves peptide
bonds in two steps
(36) . The first step is the formation of a
covalent bond between the C` atom of the scissible peptide bond and the
hydroxyl oxygen O
Therefore, the acyl-enzyme intermediate and the corresponding
tetrahedral adduct formed in the deacylation step with a single P1
residue (Arg) as the substrate component are the simplest models for
calculating enzyme-substrate interactions and investigating the nature
of the primary specificity of SPCs. Moreover, comparison of the
structures of the acyl-enzymes and the tetrahedral intermediates allows
us to investigate the stereochemistry of their mutual transformations.
Models of substrate components (consisting of a single Arg residue) for
both intermediate states of the enzymes SPC2 and SPC3 are shown in
Fig. 2
.
In the case of more complex substrate components that include
several Arg residues (e.g. Arg-Ala-Ala-Arg), we have taken
into account the same conformational parameters for the side chain of
P4 Arg as for the P1 Arg and also have calculated the angles of
rotation around the C
Molecular modeling was carried
out using the Insight and Discover graphical environments (Biosym
Technologies, Inc., San Diego). The molecular mechanics energetic
calculations utilized the force field cvff (consistent valence
force field), which was specifically derived to apply to structures of
peptides and proteins. In this approximation the formation of hydrogen
bonds is modeled as an electrostatic interaction. Electrostatic
interactions were calculated using a dielectric constant
The procedure for
minimization of the potential energy of enzyme-substrate interactions
consisted of two steps. First the arrangement of the substrate
component was optimized within the field of the rigid binding cavity of
the putative active sites of the enzymes SPC3 and SPC2, taking into
account simultaneous variations in all the above described dihedral
angles of rotation
With few exceptions the SPCs possess unique
primary specificity toward Arg residues
(19) . To account for
this our investigation of enzyme-substrate interactions began with an
analysis of the structural correspondence of the side chain of Arg (P1)
to the S1 pocket of the active sites of SPC2 and SPC3.
Minimization
of the potential energies of interaction of substrate components
(including P1 Arg) (Fig. 2) with active sites of SPC2 and SPC3
was performed on the basis of zero approximation for angles of rotation
Among the three possible values for the angle of
rotation around bond C
As
mentioned above, minimization of the potential energy of complexes has
been carried out in two steps in the field of rigid and flexible active
sites. As a result of the second step of minimization, displacements of
the positions of the C
The optimal arrangements found by
energy minimization for the side chain of Arg in the S1 subsite in the
tetrahedral intermediates formed by SPC3 and SPC2 are shown in
Fig. 3
, a and b. Angles of rotation around
bonds of both substrate components are
However, Asp
Moreover, the high degree of
structural correspondence of the S1 pocket to the side chain of Arg,
especially its guanidinium group, allows us to explain the preferential
cleavage by SPCs of peptide bonds formed by Arg residues in comparison
with Lys in P1 positions
(15, 19) , i.e. the
shorter length of the side chain of Lys does not allow formation of the
hydrogen bond with the side chain of Asp
The foregoing
conclusions do not agree with the amino acid alignment recently
proposed
(24) for furin in which the conserved Gly
It is well established that Asn
For the
correct alignment of peptide bonds undergoing cleavage by serine
proteinases, substrates must also satisfy additional secondary
specificity requirements. In subtilisin-like enzymes the main chain of
residues P1-P4 of the substrates forms a
Before consideration of the
binding of real substrates containing Arg or Lys at positions P2 and P4
it is necessary to determine the optimal packing of the main chain of
these residues. For this aim we considered the binding of a
tetrapeptide containing Arg (P1) and 3 alanines at P2-P4 under the
conditions of forming the tetrahedral intermediate. The orientation of
the side chain of the P1 Arg residue was rigidly fixed as found in the
previous step of calculation (Fig. 3a) and then the
angles of rotation,
The angles of rotation
In the
molecular model of the tetrahedral adduct of SPC3 with Ala-Ala-Ala-Arg
(Fig. 3c), it can be seen that the P4 Ala is located
exactly on top of another potential binding pocket of SPC3, the S4
locus. In the subtilisins this region is filled by bulky hydrophobic
residues (Tyr
The deduced packing of the main chain of residues P2-P4
in the crevice S2-S4 of the active sites provides a basis for
consideration of the binding of a real substrate of SPC3 having Arg at
position P4, i.e. Arg-Ala-Ala-Arg. Optimization of the
arrangement of the side chain of Arg (P4) in the binding cavity S4 was
carried out after fixation of the main chain of residues P1-P4 in the
previous step of calculation and simultaneous changes in the
conformations of the side chains both of Arg (P4) and residues
Asp
All three
negatively charged residues forming the walls of the S4 locus,
Asp
To summarize, our molecular modeling indicates that the
structure of the S4 loci of the SPCs corresponds well to the size and
shape of the side chain of Arg and provides significant electrostatic
stabilization of binding for this residue. It is possible that the
presence of Asn in position 274
(131) of SPC2 instead of Asp (as
in SPC3) explains the reduced sensitivity of PC2 to the nature of the
residue in the P4 position
(5) .
The conformational
possibilities for Lys and Arg residues at the P2 position of substrates
have been investigated by simple substitution of the P2 Ala by Arg or
Lys in the model peptide Arg-Ala-Ala-Arg in order to assess its optimal
arrangement (Fig. 3e). As a rule, in the subtilisins the
S2 loci (mainly comprised of Asp
In summary, from
the considerations we have discussed here we can conclude that the
structures of the active sites of the SPCs, SPC2 and SPC3, which were
modeled on the basis of their assumed homology with the tertiary
structures of the subtilisins, satisfy the conditions for formation of
dense energetic packing with substrates of the type
Arg-X-Lys/Arg-Arg in the acyl-enzyme and tetrahedral
intermediate states. Each of the active site subsites (S1-S4) contain
deeply located residues of Asp or Glu (primarily Asp
Residue
numbering corresponds to SPC3 (13) and subtilisin BPN (27) (in
parentheses). Numbering for this alignment of the SPCs and kexin is
given in Ref. 13.
We thank Florence Rozenfeld for her expert assistance
in the preparation of this manuscript.
ABSTRACT
INTRODUCTION
Structures of the Active Sites of Subtilisins and
SPCs
Conformational Models and Methods of
Calculation
Investigation of the Primary Substrate Specificity of
SPC2 and SPC3
Secondary Specificity of SPC2 and SPC3
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Pro
) in this loop in the subtilisins
and its absence in the SPCs. Accordingly, we have proposed a new
alignment for the amino acid sequences of the SPCs in this region. Both
SPC2 and SPC3 participate in the processing of prohormones at dibasic
cleavage sites, typically Lys-Arg or Arg-Arg. To investigate the
structural basis of the substrate specificity of these SPCs, we have
carried out molecular mechanic calculations of the optimal arrangement
and interactions of peptide substrates containing several residues of
arginine or lysine, i.e. Arg, Ala-Ala-Ala-Arg,
Arg-Ala-Ala-Arg, Arg-Ala-Arg-Arg, Arg-Ala-Lys-Arg, in the putative
active sites. Such subtilisin-based modeling has allowed us to identify
those negatively charged residues, Asp and Glu, in the S1, S2, and S4
subsites, which can directly interact with basic residues in the
substrates via formation of salt bridges and thereby contribute to the
substrate selectivity of the SPCs.
(
)(9) , PACE4 (SPC4)
(10) ,
and the prohormone convertases PC2 (SPC2)
(11, 12) ,
PC1/PC3 (SPC3)
(13, 14) , PC4 (SPC5)
(15) , and
PC5/PC6 (SPC6)
(16) .
(
)
of peptide inhibitors and/or
substrates.
-Pro
in the subtilisins),
cis-peptide bonds predominate
(32) . In the case of the
subtilisins, this cis-peptide bond forms a sharp type VI
-turn (33, .2) in the inner loop 165-171 which
serves as a floor for the active sites.
makes
close van der Waals contacts with the backbone and the side chains of
residues 152 and 153, as well as with the side chain of residue 165
(Asp in SPC2 and SPC3, ).
and Tyr
at ends of the
aforementioned loop. This packing has an energetically favored
perpendicular arrangement of the aromatic rings
(34) and is
likely to be conserved in the structures of both the subtilisins and
SPCs. In the alignment of Siezen et al.(21) , residue
167 is tyrosine in all enzymes; however, the downstream residue
(Tyr
) in the sequences of the SPCs has been aligned with
residue 172 (Pro or Asp) of the subtilisins. Such an alignment is
unlikely according to the matrix of mutual substitutions of amino acid
residues in proteins
(35) . For this reason it is difficult to
accept the latest alignment given by Siezen et al.(24) where Tyr
and Tyr
of the
subtilisins and thermitase are substituted by Gly and Ser in the SPCs.
Such substitutions would be expected to lead to a loss of the energy of
nonbonded interactions between residues of Tyr
and
Tyr
which for thermitase comprises
-7
kcal/mol. It is important that all known SPCs contain these 2 highly
conserved aromatic residues in this loop (24, ).
-Tyr
interaction
in the SPCs, we propose a new alignment for sequences 165-171 of
the subtilisins and the corresponding residues in SPC3 and SPC2
(). In this alignment a small residue (Ser) occurs in
position 325
(169)
(
)
at the bottom of the
S1 binding cavity, and the bulky side chain of Pro
(168) of the
subtilisins is substituted by the side chains of residues Asp, Ser, or
Asn, which are located outside the S1 groove in the SPCs. As a
consequence, the primary structures of the SPCs contain an inserted
residue of Ala or Thr between residues
(167) and
(168) in
the subtilisins. We propose that this additional residue in this loop
in the SPCs compensates for the loss of the cis peptide bond
of the subtilisins and promotes the formation of a new central
-turn, involving residues 320-327 of SPC3, which consists
entirely of trans-peptide bonds ().
(
)
atoms of residues(165-167) and(168-171), removed the
cis-peptide bond, and introduced a new residue, either Ala or
Thr, between the C
atoms of residues
(167) and
(168) . This residue could be accommodated if it
adopted the appropriate conformation of a classical
-turn, which
corresponds on a Ramachandran plot to the lower right square H (33;
.2). This
-turn forms a sharp reverse turn in the
mid-region of the 320-327(165-171) loop of the SPCs which
is stabilized by the hydrogen bond CO (Tyr
)
HN (Asp
) and consists entirely of
trans-peptide bonds (Fig. 1).
Figure 1:
Backbone
structures of the loop 165-171 in subtilisin (a) and the
loop 320-327 (165-171) in SPCs
(b).
Upon energy
optimization of this initial structure (see ``Conformations Models
and Methods of Calculation''), we found a new conformation for the
loop 320-327(165-171) in SPC3, which conserved effective
interactions of the aromatic side chains of residues 322 (167) and
327
(171) . The new version of this loop (Fig. 1) has been
introduced into the structures of subtilisins BPN` and Carlsberg by
means of superposition of equivalent -carbon atoms (except for
C
of the inserted residue) of the new and old loops
with a root-mean-square deviation of 0.35 Å. All the
following calculations of enzyme-substrate interactions have been
performed with SPC active site models having this new loop structure.
of the side chain of the catalytic
Ser
residue. This acyl-enzyme intermediate proceeds
through a negatively charged tetrahedral intermediate where the
nitrogen atom of the peptide bond is protonated by His
.
The second step of the reaction, deacylation, includes hydrolysis of
the acyl-enzyme by the attack of a water molecule, which is activated
also by His
, to regenerate the free enzyme and release the
product. This step also proceeds through a negatively charged
tetrahedral intermediate. The side chain of Asn
and the
NH group of Ser
take part in the stabilization of the
tetrahedral intermediate in the catalytic pathway of the subtilases
(36, 41). The only exception at this position in the SPCs is
Asp
in SPC2
(5) . However, SPC2 is optimally active
at acidic pH 5.5, suggesting that this Asp could interact with the
tetrahedral intermediate in its protonated form
(11) .
Figure 2:
Conformational parameters of the substrate
component in the complexes of SPC3 or SPC2 with Arg (P1) in
the stage of formation of the acyl-enzyme (a) and the
tetrahedral intermediate (b).
The conformational possibilities for substrate
components are determined by the dihedral angles of rotation around the
C-N and C
-C` bonds of the main chain,
(C
-N) and
(C
-C`), and
around the side chain bonds, angles
(C
-C
),
(C
-C
),
(C
-C
) and
(C
-N), of the Arg residue
(Fig. 2). In addition, the dihedral angles of rotation around the
side chain bonds of Ser
(221) [(
(C
-C
),
(C
-O
))
and around the complex or simple ester bond
(
(O
-C`) between this Ser
residue and Arg (P1)] were calculated. The determination and
description of the angles of rotation in the peptide chain corresponds
to standard nomenclature (33, 35). The angles of rotation equal zero
when the corresponding bonds in the backbone and in the side chain have
a cis orientation. For the values of bond lengths and valence
angles, the averaged parameters for peptide and ester bonds were
used
(35) . Zero approximation for the valence angles at the C`
atom of the scissible bond of the tetrahedral adduct was 109.5°.
-N and C
-C` bonds
of the main chain; i.e.
and
for residue P1,
and
for P2,
and
for P3, and
and
for P4.
=
4. This value corresponds to the microscopic dielectric constant of
polypeptides
(39) and usually is used for conformational
calculations in polar media
(37) . N- and C- ends of peptide
fragments of active sites were considered in neutral form as
N-methyl and N-acetyl amides, correspondingly. For
minimization procedures the steepest descends and conjugate gradients
have been used. Atomic coordinates of the enzymes were taken from the
Brookhaven Protein Data Base for subtilisin BPN` and subtilisin
Carlsberg (PDB codes 2 SNI and 2 SEC
(27) ). We have not had any
problems with substitutions of some amino acid residues in the active
site of subtilisin by the new residues present in SPC3 or SPC2 (Tables
I and II); the initial conformations of the side chains of
corresponding residues was conserved, with the exception of the bulky
side chain of Trp
(126) for which additional
packing calculations were performed. In the case of the new loop
320-327 (165-171) the side chain at position 324
(168) (Asp in SPC3) forms a hydrogen bond with the side chain of
residue Trp
(126) .
,
,
,
,
,
(and also
,
,
) for the P1 Arg residue
(Fig. 2). Then the potential energy of the entire
enzyme-substrate complex was minimized, allowing possible changes in
the positions of amino acid residues within the active sites.
(C
-C
),
(C
-C`), and
(C
-C
), found in
complexes of subtilisin with different inhibitors. In complexes, atom
O
of the side chain of residue Ser
(221) is turned by
120° relative to its original
position (
180°) in
the free enzymes. Thus, in the case of the complex of subtilisin
Carlsberg with eglin-c the value of angle
=
-81.6
(27) . For the residue Leu (P1) in the same complex
(C
-C
) =
-66.2°.
-C
(
) of the side chain of the P1 residue,
-60, 180 and 60°, corresponding to minima of the rotational
potential, only in the case of
-60°
can this side chain be arranged in the S1 subsite, while the rotamer
60° leads to significant hindrances with
residues on the bottom of the active site and the
180 promotes its location in the external region of the enzyme.
By following similar mutual considerations only the values in proximity
to
120° and -60° are correspondingly admissible for
(C
-O
)
and
(C
-C`). The angle
(O
-C`) was taken equal to
180°, which corresponds to the trans configuration of the
complex ester group, as is usual for acyl-enzymes (38). Initial values
for the remaining angles of the side chain of Arg,
,
, and
, corresponded to the
trans orientation of the C-C bonds (
180°), since this
side chain has a tendency to accept extended conformations in active
sites of enzymes, as for example, in trypsin
(39) .
backbone atoms did not exceed
0.3 Å relative to the initial rigid model of the active sites.
However, such small changes were enough to remove non-bonded repulsions
between the guanidinium group of the side chain of Arg (P1) and the
atoms of the walls of the S1 subsite, primarily with 3 Gly residues
(269
(127) , 308
(154) , and 321
(166) ). A similar
effect was observed in an x-ray study of the adduct of
2-phenylethaneboronic acid with subtilisin BPN`
(40) . An
expansion of the binding crevice of subtilisin by perhaps as much as
0.5 Å was interpreted as an effect induced by binding of the
aromatic ring of the inhibitor.
-70.1°,
100.4°,
-172.1°,
-50.9°,
-81.1°,
175.5°,
178.8°,
170.4° (in the
acyl-enzyme) and
-68.8°,
101.5°,
-167.8°,
-66.5°,
-69.9°,
175.1°,
179.9°,
-170.1° (in the tetrahedral intermediate). These
calculations show that the conformation of the substrate component of
the tetrahedral adduct at the deacylation step is practically
coincident with its conformation in the acyl-enzyme. It is also valid
for the arrangement of the guanidinium group of the side chain of Arg
in both intermediate states.
Figure 3:
Spatial models of the tetrahedral adducts
SPC3 with Arg (P1) (a); Arg (P1) (for SPC2
with Asp at position 310 (155)) (b); Ala-Ala-Ala-Arg
(P1-P4) (c); Arg-Ala-Ala-Arg (d);
Arg-Ala-Arg-Arg (e). The substrates and important residues of
enzymes are shown in a space-filling image.
The size and shape of the S1 pocket of
enzymes SPC3 and SPC2 exactly corresponds to the extended conformation
of the Arg side chain. The walls of the binding cavity are restricted
by 3 residues of Gly, 269 (127), 308
(154) , and
321
(166) , and the guanidinium group forms close van der Waals
contacts primarily with hydrogen atoms of C-H bonds of
these residues (Fig. 3, a and b). Due to such
dense packing the energy of nonbonded interactions of the side chain of
Arg (P1) with these three glycines is -4.5 kcal/mol. Thus, in
this case we have an example of high conformity between the structures
of the substrate and the active site S1 of the enzyme. Moreover, the P1
Arg side chain is located parallel to the peptide backbone of the
extended strand formed by residues 267-270(125-128), and
the energy of nonbonded and weak polar interactions with this strand
comprises
-10 kcal/mol. Therefore, this calculation allows
us to conclude that the nonbonded van der Waals attraction of the side
chain of Arg with the three walls formed by peptide segments of the S1
subsite comprises a significant energetic contribution to the binding
of the P1 Arg residue in SPC substrates.
(165) probably plays the most significant role in determining
the high primary specificity of the SPCs for P1 Arg residues. In the
subtilisins the bulky hydrophobic side chain of residue 165 (Val or
Ile) is surrounded by Ala
, Leu
,
Ala
, and by the salt bridge formed by Lys
and Glu
, whereas in the SPCs all of these residues
are replaced by small polar residues of Ser (Thr in the case of
Leu
), and therefore, the side chain of Asp
(165) can easily change its conformation. Such changes in the
environment of residue 320
(165) in the SPCs shows that it is an
internal rather than a surface residue of the protein. Binding of
substrates containing Arg residues induces conformational
transformations in the side chain of Asp
(165) from a state with an angle
(C
-C
) = 60° in native
subtilisin to a new state with
(C
-C
) = 180° when this
side chain forms the bottom of the S1 binding cavity and interacts
directly with the guanidinium group of P1 Arg. In the most optimal
orientation, the distance between the carboxyl oxygen of
Asp
(165) and one of the protons of
N
-H groups of the guanidinium group is 2 Å,
which is suitable for a hydrogen bond
(35) . The energy of
electrostatic interaction between Arg (P1) and Asp
(165) comprises -7.1 kcal/mol, whereas the energy of
nonbonded interactions is -1.2 kcal/mol. The hydrogen bond
between the backbone groups in SPC3 (NH
(Gly
)
O=C (Ser
)) is
also weakened due to direct interaction of the side chain of
Asp
(165) with the NH bond of
Gly
(166) .
(165) (the distance
N
(Lys)
O
(Asp) is 4.5
Å). Mutagenesis of the corresponding Asp residue in furin to Asn
(Asp
(165) in our alignment) reduced its activity
dramatically to below detectable levels
(23) . Thus, this
aspartic acid residue (320 in SPC3) clearly participates in the
recognition of scissible peptide bonds. In sum then, the triad of
glycine residues (269
(127) , 308
(154) , and
321
(166) ), in the S1 pocket of SPC2 and SPC3 provides suitable
structural complementarity for the P1 Arg side chain of substrates,
while the primary specificity of the SPCs for Arg residues is
determined by the side chain of Asp
(165) , which
is located deeply in the bottom of the S1 pocket.
residue of the subtilases is aligned with Asp in the processing
endoproteases (Asp
in SPC3). The presence of the
additional C
atom at position 166 hinders the binding
of substrates having long bulky side chains at position P1, i.e. Tyr
(41) . Moreover, the side chain at position 166 is
external, does not form hydrogen bonds with Arg (P1), and is likely to
interact with the aqueous medium. Indeed, double mutation of subtilisin
BPN` (Gly
Asp and Asn
Asp) produces a
subtilisin variant having greater specificity for dibasic pairs where
Lys occupies the P1 position, i.e. Lys Lys and Arg
Lys
(42) . However, the SPCs exhibit a strong preference for
sites containing P1 Arg residues and discriminate significantly against
Lys at this position. An exception is the cleavage of some Lys-Lys
bonds by SPC2, which may result from an alternative mechanism for
binding of the side chain of P1 Lys residues by Asp
(155) that is unique to this enzyme.
(
)
plays an
important role in catalysis by the subtilisins. Its side chain is
believed to provide one of the hydrogen bonding groups for stabilizing
the negatively charged tetrahedral intermediate in the transition
state
(36, 43) . According to our calculations of the
optimal conformation of the substrate component in the acyl-enzyme, the
carbonyl of the complex ester group forms two hydrogen bonds with amide
groups of the main chain of Ser
(221) and the side
chain of Asn
(155) in SPC3, and the corresponding
O
H distances equal 2.4 Å. In the case of the
tetrahedral intermediate (Fig. 3a) the negatively
charged oxygen of the scissible bond also forms the same hydrogen
bonds, but they now become significantly shorter, the
O
H distances in both segments
C-O
H-N equal 1.8 Å, an
optimal value for hydrogen bonds of this type
(35) . A similar
hydrogen bond can be formed with the protonated side chain of
Asp
(155) in the case of PC2
(5) (Fig. 3b). The tendency for formation of
strong hydrogen bonds appears to be maximal in the tetrahedral
intermediate. Evaluation of the energetic stabilization of the
tetrahedral intermediate by the additional electrostatic interactions
with residues Asn
(155) and Ser
(221) relative to the acyl-enzyme gives a calculated value of
-3 kcal/mol. The experimental value for the energy of
stabilization of the transition state in subtilisin catalysis is
-3.7 kcal/mol
(44) .
-strand structure with
the surface crevice (loci S2-S4) of the active sites, residues
100-103 and 125-128
(36) . Secondary specificity
requirements appear to be significantly more stringent for the SPCs
than the subtilisins in that Lys and Arg residues at P2 and P4 play an
essential role in determining substrate
specificity
(15, 20) .
and
, around bonds C
-N
and C
-C`, respectively, were optimized for each of the
main chain residues P1-P4. As an initial approximation
and
were chosen to satisfy the formation of hydrogen bonds within
the limits of 2.8-3.5 Å between the substrate component and
the peptide backbone of segments 243-246(100-103) and
267-270(125-128) of the enzymes.
. . .
found as a result of
minimization of the potential energy of the interactions of
Ala-Ala-Ala-Arg with SPC3 are
-75.5°,
-63.1°,
107.1°,
-136.9°,
148.1°,
-82.4° and
151.0°.
The network of hydrogen bonds formed by this peptide with segments
243-246 (100-103) and 267-270(125-128) of the
enzyme are practically the same as in the complex of subtilisin with
eglin
(27) . However, the hydrogen bond with the carbonyl group
of Gly
(127) is less satisfactory because the
adjacent Pro
(128) in the case of SPC2 or SPC3
hinders the formation of hydrogen bonds by this residue.
, Ile
, Leu
, and
Pro
) which enhance the binding of hydrophobic P4
residues
(45) . However, in the SPCs the corresponding residues
at positions 247
(104) and 250
(107) are Asp and Glu,
respectively (). Moreover, such changes in the S4 region
allow residue 284
(135) , which is Ala or Thr in the SPCs instead
of the bulky residues (Leu or Met) of the subtilisins, to form a new
floor for the S4 cavity. The distance between the C
atom of the P4 Ala of the substrate Ala-Ala-Ala-Arg and the
C
atom of the new residue 284
(135) (Ala)
corresponds closely to the length of the Arg side chain in extended
conformation. It is likely that the substitution of Gly
in the subtilisins by Pro
in SPCs ()
provides further orientation for the backbone of the side chain of
residue P4.
(104) , Glu
(107) ,
Asp
(131) , and Trp
(126) .
Optimal values for the angles of rotation around bonds of the side
chain of the P4 Arg are
-53.6°,
-159.6°,
161.1°,
-169.9°. Only the rotamer with the value
of
(C
-C
)
-60° was available while other angles
,
, and
corresponded to the extended
conformation (
180°) of the side chain. As in the case of the
P1 Arg, the side chain of the P4 Arg conformed well with the surface of
the S4 binding cavity. The guanidinium group of the P4 Arg made van der
Waals contacts with the side chains of residues
Asp
(104) , Glu
(107),
Ala
(135) , and Trp
(126) , and
the energy of nonbonded dispersion interactions between the side chain
of Arg (P4) and residues of the binding site S4 comprised
-11 kcal/mol. A spatial model of the binding of Arg-Ala-Ala-Arg
by SPC3 (loci S1-S4) is shown in Fig. 3d.
(104) , Glu
(107) , and
Asp
(131) , can form strong hydrogen bonds or salt
bridges with the guanidinium group of Arg (P4). This is possible when
the side chain of Asp
(104) adopts a conformation
with
(C
-C
)
180°, of Glu
(107) ,
-60°, and of
Asp
(131) ,
-160° (Fig. 3d). It is evident that
electrostatic interactions with these side chains (calculated value
-19.5 kcal/mol) participate significantly in the energy of
binding of the P4 Arg residue. However, Asp
(104) is a surface residue of the SPCs, and thus its side chain
can easily change its conformational state, while the side chains of
residues Glu
(107) and Asp
(131) are located deeply within the bottom of the S4 locus.
Binding of the P4 Arg residue of a substrate freezes their
conformations. Indeed, substitution of Asp
(104) by Asn in furin had little effect on the activity of this
enzyme
(23) . The side chain of Asp
(104) is
thus not likely to interact with the P4 Arg side chain. On the other
hand, site-directed mutation of the 2 neighboring Asp residues (272
(130) and 273
(131) ) () of furin (Asp-Asp
Ser-Gly) led to inhibition of the processing of precursors at
sites containing a P4 Arg residue (Arg-X-X-Arg)
(23) .
However, in our model the Asp
(130) side chain is
directed away from the S4 subsite. Thus, the substrate specificity of
the SPCs relative to Arg (P4) is determined mainly by internal (but not
external) carboxyl groups within the spatial structures of these
proteins.
, His
,
Leu
, and Asn
) bind small nonpolar residues
(Thr in the case of eglin c). Moreover, it is impossible to direct the
relatively long side chains of Lys or Arg into the S2 pocket of the
SPCs due to strong, nonbonded repulsions with the residues lining the
inner wall of this pocket (for example Asp
(33) ;
see ). Therefore the P2 Lys and Arg side chains can be
located only on the external surface of the active site where they may
interact with the side chains of His
(64) and
Asn
(62) . As before, the side chain of the P2 Arg
adopts an extended conformation (
180°) with an optimal value
for the
(C
-C
) angle
of 88.5. In the orientation we have deduced for the P2 Arg, the
conserved Asp
(33) of the S2 subsites of the SPCs
does not interact directly with this residue; however, it can play an
important orienting role by fixing the conformation of the side chain
of Asn
(62) such that its amide group forms
hydrogen bonds with the side chains of P2 Lys or Arg, thereby promoting
the binding of these basic residues. A stereo view of a model of the
binding of Arg-Ala-Arg-Arg is shown in Fig. 4.
Figure 4:
Stereo view of the proposed arrangement
of the substrate Arg-Ala-Arg-Arg (heavy line) in the active
site of SPC3 (in accordance with Fig.
3e).
Participation
of residue 206
(62) in processing follows from data for kexin in
which the presence of Asp instead of Asn at this position significantly
increases the preference for substrates containing P2 Lys residues,
while the nature of the P4 residue is irrelevant
(46) . It is
likely that the interaction of the side chain of Asp (62)
with the side chains of P2 Lys or Arg compensates for the loss of
effective S4 binding in kexin. In contrast, it has been shown that
sites of the type Arg-X-X-Arg are effectively processed by
both SPC3
(15, 19) and furin
(20) . Thus, the
processing by these SPCs requires cleavage sites containing primarily a
P1 Arg residue and a second basic residue at either P2 or P4 such that
the loss of effective binding in the S2 subsite is compensated by
binding in the S4 subsite and vice versa.
(165) in the S1 subsite, Asp
(33) in the S2
subsite, and Glu
(107) and Asp
(131)
in the S4 subsite), and these determine the high specificity of these
enzymes toward substrates having basic amino acid residues at positions
P1, P2, and P4. It appears that all the SPCs share this fundamentally
very similar specificity for P1, P2, and P4 basic residues but their
selectivity for individual substrates is dictated in part by additional
features (possibly residues at P5, P6, P1`, and P2`), as well as by the
more acidic pH optima of the prohormone convertases, SPC2 and SPC3.
Table:
Alignment of amino acid residues in active site
regions of subtilisins BPM` and Carlsberg and SPCs (21)
Table:
New proposal for alignment of
amino acid residues in SPCs (sequences 319-329 of SPC3 and
164-175 in subtilisin BPN`)
Thr of subtilisin BPN` (Q.
Gong, unpublished data). Western blot analysis of the culture medium of
the mutant revealed the absence of mature processed subtilisin BPN`.
Therefore, this single mutation may interfere with the formation of a
cis-peptide bond and disrupt the folding pathway for this
subtilisin. This result provides evidence that Pro
plays
an important role in the self-organization of the subtilisins and that
the tertiary structures of loops 165-171 in the subtilisins and
320-327 in SPCs differ significantly. Moreover, in accordance
with our proposal the mutant Gly
Asp also was not active,
indicating that the alignment of the position 169 (324) as given in
Ref. 21 is not acceptable.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.