Báo cáo khoa học: Crystal structure of human cystatin C stabilized against amyloid formation pdf

In all crystal structures of cystatin C studied to date, the protein has been found to form 3D domain-swapped dimers, created through a confor-mational change of a b-hairpin loop, L1, fr

amyloid formation

Robert Kolodziejczyk1, Karolina Michalska1, Alejandra Hernandez-Santoyo1,2, Maria Wahlbom3, Anders Grubb3 and Mariusz Jaskolski1,4

1 Faculty of Chemistry, Department of Crystallography, A Mickiewicz University, Poznan, Poland

2 Instituto de Quı´mica, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria, Mexico

3 Department of Clinical Chemistry, Lund University, Sweden

4 Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

Introduction

The cystatin superfamily of cysteine protease inhibitors

is subdivided, according to structural features and

presence in the intracellular, extracellular or

intravas-cular space, into three families: 1 (stefins), 2 (cystatins),

and 3 (kininogens) [1–3] Human cystatin C (HCC),

a member of the family 2 cystatins, is an inhibitor of

papain-like (C1) and legumain-like (C13) cysteine

pro-teases [4] The inhibition of C1 propro-teases is especially

potent, with dissociation constants (in the femtomolar

range) [3] representing the strongest competitive inhibi-tion known in biochemistry HCC is found in all body fluids, with particularly high concentrations in the cerebrospinal fluid [5], and its function is to regulate the activity of cysteine proteases, either released from lysosomes of dying or damaged cells [6], or originating from microbial invasion [7] In common with other family 2 cystatins [1,4], the 120 residue polypeptide chain of HCC is expected to fold as a monomer with

Keywords

amyloid; crystal twinning; cysteine protease

inhibitor; protein engineering; 3D domain

swapping

Correspondence

M Jaskolski, Faculty of Chemistry,

Department of Crystallography, A.

Mickiewicz University, Grunwaldzka 6,

60-780 Poznan, Poland

Fax +48 61 8291505

Tel: +48 61 8291274

E-mail: mariuszj@amu.edu.pl

Database

The atomic coordinates and structure

factors have been deposited in the Protein

Data Bank under the accession code 3GAX

(Received 4 January 2010, revised 25

January 2010, accepted 27 January 2010)

doi:10.1111/j.1742-4658.2010.07596.x

Human cystatin C (HCC) is a family 2 cystatin inhibitor of papain-like (C1) and legumain-related (C13) cysteine proteases In pathophysiological processes, the nature of which is not understood, HCC is codeposited in the amyloid plaques of Alzheimer’s disease or Down’s syndrome The amy-loidogenic properties of HCC are greatly increased in a naturally occurring L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult life In all crystal structures of cystatin C studied to date, the protein has been found to form 3D domain-swapped dimers, created through a confor-mational change of a b-hairpin loop, L1, from the papain-binding epitope

We have created monomer-stabilized human cystatin C, with an engineered disulfide bond (L47C)–(G69C) between the structural elements that become separated upon domain swapping The mutant has drastically reduced dimerization and fibril formation properties, but its inhibition of papain is unaltered The structure confirms the success of the protein engineering experiment to abolish 3D domain swapping and, in consequence, amyloid fibril formation It illustrates for the first time the fold of monomeric cysta-tin C and allows verification of earlier predictions based on the domain-swapped forms and on the structure of chicken cystatin Importantly, the structure defines the so-far unknown conformation of loop L1, which is essential for the inhibition of papain-like cysteine proteases

Abbreviations

AS, appending structure; HCC, human cystatin C; HCCAA, hereditary cystatin C amyloid angiopathy; HCC-stab, monomer-stabilized human cystatin C; PDB, Protein Data Bank.

two well-conserved disulfide bridges (Cys73–Cys83 and

Cys97–Cys117) in the C-terminal half of the sequence

[8,9] By analogy with enzyme complexes of family 1

cystatins [10–12], and chicken cystatin docked in the

active site of papain [13], the inhibitory epitope of

HCC has been postulated to include the N-terminal

peptide Ser1–Val10, and two b-hairpin loops, L1,

con-taining a signature motif QxVxG(55–59), and L2, with

a characteristic Pro105-Trp106 sequence, all aligned in

a wedge-like fashion at one side of the molecule [4]

The need to define a precise enzyme-binding motif

was dictated by the idea of using it as a molecular

tem-plate in the design of efficient small molecule inhibitors,

targeting cysteine proteases involved in

tissue-degenera-tive diseases, such as osteoporosis or paradontosis [14],

or produced by highly virulent strains of bacteria and

viruses [15–18] To this end, we have carried out

numerous crystallization experiments on papain–HCC

mixtures However, single crystals of a papain–HCC

complex could never be obtained, as the incubated

samples invariably undergo proteolytic degradation,

probably because of impurities present in commercially

available papain samples Also, despite many years of

effort, crystallization of monomeric HCC has not been

achieved Instead, a number of crystallization

condi-tions have invariably led to crystals being formed from

HCC dimers, arising as a consequence of 3D domain

swapping [19–21], a phenomenon in which the

mono-meric fold is re-created in an oligomono-meric context, i.e

from fragments of the polypeptide chain contributed

by different molecules [22] In a closed 3D domain-swapped dimer, the protein fold is essentially as in the monomeric form, except in a hinge element, which has

a drastically changed conformation, allowing the protein to partially unfold and form a mutual grip with a similarly unfolded partner In the HCC dimer, the hinge element is loop L1, which assumes an extended conformation engaged in a long intermolecu-lar b-sheet, and the N-terminal fragment of the mole-cule (b1–a–b2) is anchored in the domain-swapped partner molecule (Fig 1B) This unexpected difficulty has opened a completely new aspect of HCC research, connected with a naturally occurring L68Q variant of HCC, whose extremely high propensity for dimeriza-tion and aggregadimeriza-tion leads to amyloid deposits in cerebral vasculature in a lethal disease known as hereditary cystatin C amyloid angiopathy [23,24] The discovery of a domain-swapped HCC structure [19] provided the first experimental evidence of 3D domain swapping in an amyloidogenic protein, and has rekindled interest in this phenomenon as a possible mechanism of amyloid formation [25,26] Since then, 3D domain swapping has been demonstrated in two other amyloidogenic proteins, namely the prion protein [27] and b-microglobulin [28] The interest in HCC, however, has not declined, partly because the dimeric protein has been crystallized in several forms [29], including a polymorph in which the domain-swapped molecules have aggregated to build an infinite structure with all b-chains in a perpendicular orientation relative

B A

Fig 1 The fold of HCC (A) Molecule A of HCC-stab1 (this work) folded as a monomer The papain-binding epitope is formed by the N-ter-minus, loop L1, and loop L2 The AS is an irregular appending structure at a ‘back side’ loop system harboring a potential legumain-binding site The dashed line represents a fragment of the HCC-stab1 backbone that is not visible in the electron density map of molecule A L47C and G69C denote the Cys mutation sites in HCC-stab1, introduced to provide a covalent disulfide link between strands b2 and b3 (B) For comparison, the 3D domain-swapped dimer of wild-type HCC is shown (PDB code: 1G96) It has a folding domain similar to the monomeric HCC-stab1, but composed of two molecules (blue and green) All of the structural elements of the monomeric fold are preserved, except for loop L1, which is transformed to an extended conformation.

to a common direction [21], as required by the cross-b

canon [30] of amyloid fibril architecture Although

the present view of amyloid aggregation is more

complex [31], the interest in 3D domain swapping

remains high

Until recently, the available experimental data were

insufficient to decide between two models relating 3D

domain swapping and amyloid structure In one of

them, 3D domain swapping would operate in a

propa-gated, open-ended fashion linking all the molecules

into a runaway structure According to the other view,

3D domain swapping would only be necessary for the

formation of dimeric building blocks, which would

aggregate to form fibrils using a different mechanism

Two recently reported experiments have demonstrated

that, at least in the amyloid fibrils of T7 endonuclease

[32] and of HCC [33], 3D domain swapping in the

propagated mode is taking place

A related research interest is focused on ways to

pre-vent oligomerization and aggregation of amyloidogenic

proteins In one study, catalytic amounts of antibody

or enzymatically inactive papain prevented

dimeriza-tion of wild-type and L68Q HCC [34] Also, it was

possible to inhibit dimerization, oligomerization and

amyloid formation of HCC by site-directed

mutagene-sis of its sequence Specifically, under the assumption

that in a 3D domain-swapped dimer the organization

of the structural elements closely resembles the

mono-meric fold, the dimono-meric structures of HCC were

ana-lyzed in order to identify places where a covalent

crosslink would tether those structural elements of the

monomer that undergo separation during domain

swapping Thus, pairs of juxtaposed Cys residues were

introduced into the HCC sequence, with the

expecta-tion that their connecexpecta-tion through a disulfide bond

would provide the necessary crosslinkage [34] Two

monomer-stabilizing disulfide bridges were introduced

in this manner, between strands b2 and b3

(monomer-stabilized HCC-stab1 double mutant L47C⁄ G69C) or

between the a-helix and strand b5 (HCC-stab2 double

mutant P29C⁄ M110C)

In this work, we have crystallized the HCC-stab1

mutant and determined its 3D structure at 1.7 A˚

resolution The structure confirms the success of the

protein engineering experiment to abrogate 3D domain

swapping, and demonstrates for the first time HCC

folded as a monomeric protein It allows verification

of the earlier predictions based on the

domain-swapped form or on the crystal structure of chicken

cystatin [13,35,36] Importantly, the structure of

HCC-stab1 defines the so-far unknown conformation of

loop L1, an element that is essential for the inhibition of

papain-like cysteine proteases

Results and Discussion

Structure solution and refinement Initial analysis of the diffraction pattern of poorly dif-fracting HCC-stab1 crystals (2.6 A˚) suggested that they have hexagonal symmetry, with 12 protein molecules in the unit cell A successful run of molecular replacement with chicken cystatin [Protein Data Bank (PDB) code: 1CEW] as a model confirmed the correctness of the P61 space group and, indeed, revealed two HCC-stab1 mole-cules in the asymmetric unit A complete model with correct sequence was obtained after several rounds of manual rebuilding and maximum likelihood refinement Although the atomic model agreed well with electron density maps, the refinement was characterized by high R-factors Re-examination of the data revealed hemi-hedral twinning with a twin law (h, – h – k, – l) The same kind of twinning was detected in a new dataset, with a resolution of 1.7 A˚, that was collected for a crys-tal from a different cryscrys-tallization trial The subsequent refinement, carried out in refmac5 [37] with the appro-priate twin option, used the new data and a set of Rfree reflections selected in pairs according to the twin law The final value of the twinning fraction, a = 0.452, indicates almost perfect twinning The refinement con-verged with R and Rfreeof 0.138 and 0.167, respectively, and with other parameters as listed in Table 1

Description of monomeric HCC structure Overall fold

The two HCC-stab1 molecules (A and B) in the asym-metric unit display the canonical cystatin fold, (N)–b1– a–b2–L1–b3–AS–b4–L2–b5–(C), with a five-stranded antiparallel b-sheet gripped around a long a-helix (Fig 1) The AS is a broad irregular ‘appending struc-ture’ positioned at the opposite end of the b-sheet relative to the N-terminus⁄ loop L1 ⁄ loop L2 edge, which is the papain-binding epitope b1 is the shortest element of the five-stranded antiparallel b-sheet, com-prising only two residues In both molecules, the first

11 residues are disordered and not visible in electron density maps Two AS residues, different in each mole-cule, are also disordered: Pro78-Leu79 in A and Leu80-Asp81 in B Some of the disulfide bridges are evidently partially broken, an effect that can be attributed to radiation damage

b-Bulges The fold of monomeric cystatin C contains four b-bulges, which endow the molecular b-sheet with very

strong curvature Three of the b-bulges are classified

as antiparallel classic type [38], and the residues at

which the pattern of hydrogen bonds is broken are

Asp65 (at b2–b3), Glu67 (b2–b3), and Gln100 (b4–b5)

The fourth b-bulge, representing a special antiparallel

type, is created by His43 (b2–b3) An identical system

of deviations from regular b-sheet geometry exists in

the HCC dimers with 3D swapped domains, as well as

in chicken cystatin

b-Bulges, which disrupt the continuity of

b-interac-tions, are considered to be a strategy to avoid protein

aggregation through intermolecular b-sheet extension

[39,40] For this reason, b-bulges are desired at

edge-forming (terminal) b-strands, but are not very common

in the b-sheet interior However, the situation in HCC

is quite the opposite Three of the b-bulges are located

exactly within the inner b-sheet elements, namely at

the b2–b3 junction It is very intriguing to note that it

is this very junction that gets separated during the 3D

domain-swapping event in HCC It is therefore very

likely that the b-bulges destabilize the HCC b-sheet in

this region, thus promoting partial protein unfolding

and, eventually, oligomerization and higher-order

aggregation

The disulfide bridges The electron density map provides clear evidence of the existence of the engineered Cys47-Cys69 disulfide bond (Fig 2), introduced to covalently link strands b2 and b3 of one monomer A comparison with the structures

of 3D domain-swapped HCC (e.g PDB code: 1G69) (Fig 1B) or monomeric chicken cystatin (PDB code: 1CEW) shows that the presence of this extra bridge does not perturb the conformation of the main chain in the region of these point mutations This is possible because the side chain of Cys47 fills the space of the native Leu47, and Cys69, replacing a Gly, occupies the empty space close to the Ca atom Considering that the rigid fragments (i.e the b-sheet and a-helix) of 1G69 and 1CEW agree very well with the backbone trace of the analogous elements of HCC-stab1, we may assume that HCC-stab1 correctly represents the structure of the native HCC monomer

However, it became clear in the course of the refine-ment that the new disulfide bond had to be partially broken to fit the 2Fo– Fc electron density map con-toured at the 1.0r level Disruption of disulfide bonds

is a common effect observed in protein crystals exposed

to intense X-ray radiation, and is caused by the reduc-ing effect of free electrons generated in the ionization events Consequently, the Cys47–Cys69 bond has been modeled with partial occupancy (40% in A, and 60%

in B), and the remaining occupancy has been allocated

to free -SH groups according to indications from an

Fo–Fcmap contoured at the 3.0r level As in the preli-minary biochemical experiments (including gel filtra-tion) there was no indication of dimerization, it must

be concluded that the disruption of the Cys47–Cys69

Table 1 Data collection and refinement statistics.

Data collection

Cell dimensions (A ˚ ) a = 76.26, c = 97.57

Refinement

Reflections work ⁄ test set 34 199 ⁄ 1037

No of protein ⁄ water atoms 2020 ⁄ 180

<B-factors> protein ⁄ water (A˚ 2 ) 13.5 ⁄ 20.4

Rmsd from ideal

Ramachandran statistics of u ⁄ w angles (%)

a

Values in parentheses are for the highest-resolution shell.

Fig 2 2F o –F c electron density map contoured at the 1.0r level, in the region of the L47C ⁄ G69C mutation in molecule A, illustrating the partial disruption of the disulfide bond.

bond has occurred during the X-ray exposure of the

crystal Another ionizing radiation-driven disruption of

a disulfide bond was found at the Cys73–Cys83 bridge

of molecule A, which was modeled with 50%

occu-pancy In molecule B, this bond is intact The second

native disulfide bond, Cys97–Cys117, is intact in both

molecules The above observations illustrate the fact

that ionization-induced disruption of a disulfide bond

depends on its accessibility in both intramolecular

terms and in the crystal packing context

The new disulfide bond is right-handed, with C–S–

S–C torsion angles (v3) of 74.2 and 75.2 in

mole-cules A and B, respectively The remaining two, native,

disulfide bonds in molecules A and B have the

corresponding torsion angles of )89.0 and )97.9 for

Cys73–Cys83 and 114.7 and 114.5 for Cys97–

Cys117, thus defining them as left-handed and

right-handed, respectively A survey of these bridges in the

HCC dimers shows that the C-terminal Cys97–Cys117

bond has a rigid right-handed form, whereas the

Cys73–Cys83 bond, found in the AS region, has a

variable configuration

Conformation of loop L1

The present study reveals, for the first time, the

struc-ture of intact loop L1 of HCC, which structurally

comprises VAG(57–59) However, when referring to

loop L1 as part of the inhibitory epitope, one has to

include two additional residues at the C-terminal end

of strand b2 In this article, we follow the standard L1

nomenclature [4], referring to the QIVAG(55–59)

pen-tapeptide The electron density in the L1 area of

mole-cules A and B is of very high quality, allowing for

modeling of the backbone and side chain

conforma-tions without ambiguity (Fig 3) The VAG(57–59)

triplet can be classified as an inverse c-turn [41,42],

with a hydrogen bond between residues i and i + 2,

and i + 1 backbone dihedral angles of )70.4 and

)89.0 (u) and 69.1 and 74.4 (w) in molecules A and

B, respectively Of all the L1 amino acids, Val57 shows

the highest deformation, with backbone torsion angles

(u⁄ w) that locate it in the lower left quadrant of the

Ramachandran plot, in the additionally allowed

()112.9 ⁄ )133.4, molecule B) or generously allowed

()130.9 ⁄ )147.4, molecule A) regions

In chicken cystatin (PDB code: 1CEW), which is the

closest structural analog available for comparison,

most of the L1 residues have torsion angles in the

pre-ferred regions, except Ser56, which is located at the

apex of the loop in a position occupied in HCC-stab1

by Ala58, and which is an evident outlier

(u =)176.7, w = )56.1) This anomaly is very

diffi-cult to explain, as the two inhibitors have similar affin-ities for the cognate enzymes It is interesting to note that this conformational dissimilarity is coupled with a quite different chemical character of the residue in the apex position of loop L1 (Ala in HCC, and Ser in chicken cystatin) In the structure of stefin B in com-plex with papain (PDB code: 1STF), the L1 fragment [QVVAG(53–57)] has no outliers in the Ramachandran plot The residue in question, namely Val55, which is equivalent to Val57 in the HCC sequence, adopts a b-type conformation with u⁄ w values ()118.7 ⁄ )148.6) very close to those found in HCC-stab1 Structural environment of Leu68

Leu68 is of particular importance because its naturally occurring mutation to Gln, endemic to the Icelandic population, results in the creation of a highly amyloi-dogenic form of HCC Leu68 is located at the end of strand b3 of the b-sheet, just before the polypeptide chain enters the poorly structured and

conformational-ly variable AS The Leu68 side chain protrudes from the concave part of the b-sheet towards the interface with the a-helix The side chains of Val31 and Tyr34 (from the a-helix), Val66 (b3), Phe99 (b4), and Cys97– Cys117 (b4–b5), and the backbone of the SRA(44–46) segment, form a hydrophobic pocket in which the side chain of Leu68 is nearly ideally nested

Until now, the structural consequences of the L68Q mutation for the stability of monomeric HCC could only be inferred from the structures of 3D domain-swapped dimers of HCC The present structure pro-vides the first possibility of visualizing the Leu68 side chain in its true monomeric context Comparison with the Leu68 environment reported in the dimeric

struc-Fig 3 2Fo–Fcelectron density map contoured at 1.0r in the region

of loop L1 of molecule B.

tures shows an almost identical arrangement,

confirm-ing that the earlier analyses, usconfirm-ing the dimeric

struc-ture as a template, were essentially correct [19] The

increased size and incompatible chemical character of

a Gln side chain at position 68 would result in

repul-sive interactions destabilizing the a–b interface and

leading to an increased likelihood of a partial

unfold-ing process, in which these two main structural

ele-ments (i.e the a-helix and the b-sheet) would separate,

thus promoting oligomerization, aggregation and,

pos-sibly, fibril formation

The AS

The edge of the HCC molecule containing loop a⁄ b2

and the AS has been reported to harbor a site that is

important for the inhibition of legumain [4] The AS

itself spans 26 residues, from Cys69 to Lys94, and has

an irregular conformation The AS is located at the

opposite end of the b-sheet to the N-terminus⁄ L1 ⁄ L2

epitope (Fig 1A), so there should be no interference

between papain and legumain binding by HCC

Con-sistent with this conclusion is the observation that the

3D domain-swapped dimer of HCC shows unaltered

inhibition of legumain, whereas it is incapable of

papain binding, because of the absence of the L1

ele-ment of the recognition site [4] The backbones of

mol-ecules A and B follow the same AS trace,

complementing each other in the regions with gaps

(Fig 4A) Moreover, the conformation found in the

present structure is very similar to the backbone traces

reported in all of the dimeric structures of HCC and in

monomeric cystatin D [43] (Fig 4B) This

conforma-tion is, however, different from the models proposed for chicken cystatin or for human cystatin F [44], in which, respectively, a prominent a-helix or a helical coil is present (Fig 4C)

Crystal packing and molecular interactions The HCC-stab1 molecules A and B in the asymmetric unit form crystal packing interactions leading to their association into two types of easily recognizable assem-blies In the most conspicuous contact, molecules A and B associate via their AS loops (Fig 5), burying about 920 A˚2 of the molecular surface of each partner

in a network of interactions that includes several van der Waals contacts and four hydrogen bonds (Table 2) The two molecules are related by an almost perfect noncrystallographic two-fold axis along [1 1 0] The second mode of association buries a much smaller surface area (about 460 A˚2 per monomer), but it is important because it illustrates the propensity of HCC

to undergo intermolecular b-interactions at the edge

of the molecular b-sheet In this pairing scheme, molecule A interacts with a crystallographic copy of molecule B¢ (– y, x – y, z + 1 ⁄ 3) via a pseudo-two-fold rotation coupled with a 3 A˚ translation (Fig 5) These two molecules are linked via four main chain– main chain hydrogen bonds (Table 2) to form an inter-molecular parallel b-sheet, utilizing their b5 elements Such a mode of interaction has already been observed

in the tetragonal crystal structure of dimeric HCC (PDB code: 1TIJ) [21] Two additional hydrogen bonds involving side chains support these A–B¢ interactions The noncrystallographic symmetry axis relating the

A

C

B

Fig 4 Superposition of HCC-stab1 and

related proteins (A) The HCC-stab1

mole-cules A (red) and B (blue) superpose almost

perfectly Their AS regions (orange and

marine, respectively) complement each

other in the regions with gaps (B) Structural

alignment of HCC-stab1 (molecule A, red)

with the folding units of 3D

domain-swapped HCC molecules (1G96, orange;

1TIJ, violet) and with cystatin D monomer

(1RN7, blue) (C) Superposition of

HCC-stab1 (molecule A, red) with chicken

cystatin (1CEW, orange) and with human

cystatin F (2CH9, blue).

two molecules is aligned with the [210] direction The

translational component of this noncrystallographic

symmetry is necessary to bring the parallel b-sheet

interactions into register (Fig 5) It should be noted

that crystallographic symmetry requires both types of

dimer to sit on the same axis, so none of the diagonal

directions (related by the 31 axis) is close to a perfect

dyad Combination of the crystallographic 61axis with

the diagonal noncrystallographic dyads leads to

additional noncrystallographic two-fold axes along the

x and y directions The molecules related by this

(nearly perfect) operation present an additional scheme

of intermolecular interactions Specifically, there are

four hydrogen bonds between molecules A and B¢¢

(y – 1, – x + y, z + 5⁄ 6) at an interface formed by

loop b1⁄ a and the N-terminus of the a-helix The

con-tact area is about 220 A˚2 per molecule The

crystallo-graphic symmetry brings into contact molecules B and

B¢¢ (with the participation of loop L2), with only two

hydrogen bonds but many van der Waals contacts and

a buried surface area of about 450 A˚2 per monomer

Similar interactions are found between molecules A and A¢¢

The A–B ‘dimer’ propagates along the 31axis, utiliz-ing alternatutiliz-ing AS–AS and b5–b5 interactions, so there

is no indication of oligomeric assembly involving end-less b-sheet formation along the crystal z-axis, as observed in some of the dimeric HCC crystal struc-tures [21] The exposed b-chains at the opposite edge

of the b-sheet (b1 and b2) do not participate in any type of intermolecular b–b interaction, so no combina-tion of symmetry operacombina-tions, real or pseudo, could lead to such an assembly

It is of note that the pseudosymmetric dyad along [100] coincides with the two-fold axis relating the twin domains, which probably promotes the prevalence and character (a about 0.5) of the twinning phenomenon observed for these HCC-stab1 crystals [45]

Comparison with other cystatin models The overall fold of HCC-stab1 shows no significant differences when compared to the available models of type 2 cystatins, such as chicken cystatin (PDB code: 1CEW), cystatin D (PDB code: 1RN7), or cystatin F (PDB code: 2CH9) In addition, it is possible to compare the monomeric fold of HCC-stab1 with the complete folding units ‘extracted’ from the 3D domain-swapped dimers of HCC, namely 1G96 (one copy in the asymmetric unit), 1TIJ (two copies), and 1R4C (eight copies) In all cases, the main differences are within the AS, and concern, for example, the pres-ence of a-helical segments in 1CEW and 2CH9 (Fig 4C) When the AS fragment is excluded from the alignment, the Ca superposition becomes much closer, with, for instance, an rmsd value of 0.70 A˚ for 1G96

On the other hand, type 1 cystatin models, such as the domain-swapped stefin B (PDB code: 2OCT), and monomeric stefin A in complex with cathepsin H (PDB code: 1NB5) or stefin B in complex with papain (PDB code: 1STF), show very significant deviations from HCC-stab1, mainly in the course of the a-helix (which, in type 1 cystatins, is significantly bent), in the positioning and curvature of the b-sheet, and in the

AS, which is essentially absent in the stefin structure The results of least-squares superpositions of the HCC-stab1 molecule A and other cystatin and stefin models are summarized in Table 3

Manual docking of monomeric cystatin C in the active site of papain

The structural elements of cystatins and stefins that function as epitopes in the inhibitory interactions with

Fig 5 Crystal packing of HCC-stab1 Molecules A (red) and B

(blue) in the asymmetric unit are related by a noncrystallographic

two-fold axis parallel to [1 10], here shown (lens symbol) in

perpen-dicular orientation to the plane of the drawing The yellow (B¢) and

green (A¢) molecules are crystallographic copies (6 1 rotation along

c) of molecules B and A, respectively The components of the

(equivalent) A–B¢ and B–A¢ pairs are related by noncrystallographic

symmetry involving a two-fold axis (along [210] and [120],

respec-tively), shown as an arrow between strands b5 of the

intermolecu-lar b-sheets, and an additional 3 A ˚ translation.

their target enzymes are loops L1 and L2 and the

N-terminus [4] In cystatin C, the inhibitory part of

loop L1 has the sequence QIVAG(55–59), whereas the

most important residues from loop L2 are Pro105 and

Trp106 In an unrelated family of cysteine protease

inhibitors, represented by chagasin [46–48], the

enzyme-blocking epitope is formed by loops L4, L2

and L6, which correspond to the N-terminus⁄ L1 ⁄ L2

elements of cystatins, in that order Loop L6 contains

conserved Pro and Trp⁄ Phe residues at positions that

are equivalent to Pro105 and Trp106 in the HCC

sequence

The exact modes of cysteine protease–inhibitor inter-actions have been described, for example, for stefin B [10] (PDB code: 1STF) and chagasin [48] (PDB code: 3E1Z) in their complexes with papain These two struc-tures can be used as templates for tentative modeling of HCC-stab1–papain interactions As a first attempt, the HCC-stab1 molecule was superposed onto stefin B from the 1STF complex, using a secondary structure matching approach; this showed that the two molecules

do not align very well, with an overall Ca rmsd as high

as 2.51 A˚ Moreover, such an overall superposition leads to significant discrepancies in the epitope regions,

Table 2 Interfaces formed by pairs of HCC-stab1 molecules through crystal contacts.

Interface

Hydrogen bonds Secondary struture chID:res (atom) – (atom) res:chID secondary structure Distance (A ˚ )

A ⁄ B, B ⁄ B’’ or A ⁄ A’’

Surface area (A˚2) Rotation ()

Direction cosines of rotation axis

AS A:N82 (N d1 ) – (O d2 ) D28:B a; 3.09 a

b5 A:S115 (O) – (N) S115:B b5; 2.87 b5 A:C117 (O) – (N) C117:B b5; 2.98 b5 A:D119 (O d1 ) – (N) D119:B b5; 2.73

b1 ⁄ a A:E19 (O e1

a A:G22 (N) – (O e1 ) E19:B b1 ⁄ a; 2.95

)1.000

)1.000

a The side-chain amide of N82 has been flipped, as it evidently has the wrong rotation in the PDB entry 3GAX.

Table 3 Rmsd values (A ˚ ) between Ca atoms of structurally aligned cystatin models, defined by their PDB codes For the 3D domain-swapped dimers of HCC, the superposition is calculated for one half of the dimer, corresponding to a cystatin folding unit and containing res-idues 1–56 from one monomer (A) and 60–120 from the complementary chain (B) Explanation of PDB codes: 1G96, two-fold symmetric 3D domain-swapped HCC dimer [19]; 1R4C, 3D domain-swapped dimer of N-truncated HCC [20]; 1TIJ, 3D domain-swapped HCC dimer [21]; 1RN7, monomeric human cystatin D [43]; 1CEW, monomeric chicken cystatin [13]; 2CH9, monomeric human cystatin F [44]; 1STF, stefin B from papain complex [10]; 1NB5, stefin A from cathepsin H complex [11].

a Residues 69–94, forming the AS loop, have been omitted from the calculations.

e.g a large deviation in the Ca traces of loop L2 Thus,

in the next modeling experiment, it was assumed that

optimal fit in loops L1 and L2 should be a priority,

and Gln55–Gly59 and Val104–Gln107 of HCC-stab1

were superposed onto their equivalents in stefin B This

comparison, however, indicated that the coordinates of

loops L2 do not agree with each other, as illustrated by

the relative positions of the Ca atom of Pro105, which

differ by 1.77 A˚ in the two structures (Fig 6) Also,

forcing loops L2 to superpose worsens the alignment of

loops L1, which in a simple L1⁄ L1 superposition

over-lap almost perfectly (rmsd of 0.13 A˚) These results

clearly show that loop L2 is different in HCC and

ste-fin B One reason for this variability may be the lack of

sequence conservation, i.e the fact that the VPWQ

motif of HCC is replaced by LPHE in stefin B An

additional factor may be the involvement of loop L2 of

both molecules in the HCC-stab1 structure in similar

packing interactions, which may constrain it in a

non-native conformation The third possibility is, of course,

that the conformation of the enzyme-binding epitope,

in particular the relative disposition of loops L1 and

L2, undergoes an induced-fit adaptation on enzyme

complex formation The first hypothesis could be partly

tested using an HCC–chagasin alignment, because chagasin contains a Trp in a position equivalent to the Trp106 site of HCC Such a comparison would not be possible by direct HCC–chagasin superposition, owing

to the completely different folds of the proteins It could be achieved, however, via an intermediate align-ment of the papain components of the chagasin and stefin B complexes Then, when loop L2 from the above L1⁄ L2 superposition of HCC-stab1 and stefin B

is analyzed, it is noted that the positions of Pro105 and Trp106 match those of their chagasin counterparts much better than their equivalents in stefin B (Fig 6) Despite this improvement, Trp106 of HCC-stab1 still clashes with the Gln142 residue of papain, but this obstacle could be avoided by slight conformational adaptations of loop L2 A minor rearrangement may also be required to bring Pro105 into an optimal posi-tion with respect to Leu143 of papain, and to optimize the p-type interactions between Trp106 and the cluster

of aromatic residues of papain that cap Asn175, the last residue of the catalytic triad [46] In the current model of the HCC-stab1–papain complex, the interac-tion interface buries 564 A˚2 and 530 A˚2 of solvent-accessible surfaces of the interacting partners

The interface between papain and loop L1 of HCC-stab1 seems to be dominated by hydrophobic interac-tions, and thus resembles the situation in the stefin B complex rather than in the chagasin complex No direct interference with the Cys25-His159-Asn175 catalytic triad of papain, as observed for the chagasin–papain complex [48], is predicted from the current model The hypothetical enzyme-binding mode of the N-terminus of HCC could not be analyzed, owing to the absence of this element in the crystallographic model of HCC-stab1 However, the backbone trace near the visible beginning of the HCC-stab1 molecules follows closely the equivalent fragment of stefin B, sug-gesting that its conformation in these two proteins could be similar By analogy with other enzyme com-plex structures, the N-terminal peptide of HCC-stab1 would be expected to form b-sheet interactions with the enzyme

The above comparisons lead to the conclusion that the predicted interactions of HCC with its target enzyme, based on the structure of the current HCC-stab1 model, are compatible with various observations reported for complexes with other inhibitors However,

it is not possible to build, by simple target-based dock-ing, a model that would explain all aspects of the inhibitory interactions Therefore, a reliable description

of HCC–papain interactions will require an indepen-dent experimental study Work in this direction is in progress

Fig 6 The L1 and L2 epitope of HCC-stab1 docked into the active

site of papain The gray color represents papain from the complex

with chagasin (PDB code: 3E1Z) The papain active site residues

are shown as ball-and-stick models connected by hydrogen bonds

(red dashed lines) The key residues from the L1 and L2 epitope of

HCC-stab1 (red), as well as their counterparts in stefin B (yellow)

and chagasin (cyan), are shown in stick representation Papain

resi-dues important for interactions with loop L2 are also shown as

sticks Labels referring to the inhibitor component correspond to

the HCC sequence This overlay was constructed by first

superpos-ing loops L1 and L2 of HCC-stab1 (molecule A) on the

correspond-ing epitope of stefin B in its complex with papain (PDB code:

1STF) Next, the papain components of the 1STF and 3E1Z

com-plexes were superposed to bring the coordinates of chagasin into

the same system of reference.

Experimental procedures

Protein expression and purification

A variant of HCC with two Cys mutations introduced at

Leu47 and Gly69 was expressed in Escherichia coli MC1061

and purified using a modified procedure of Nilsson et al

[34] Briefly, expression was induced at 42C for 3 h when

D600 nm was approximately 5 The protein harvested from

the periplasmic space was purified by anion exchange

chro-matography using Q-Sepharose and a buffer containing

20 mm ethanolamine (pH 9.0) and 1 mm benzamidinium

chloride After concentration by ultrafiltration, the sample

was subjected to size exclusion chromatography using an

Amersham Biosciences FPLC Superdex HR 75 column,

equilibrated with 10 mm sodium phosphate buffer (pH 7.4),

140 mm NaCl, 3 mm KCl, and 1 mm benzamidinium

chlo-ride In the last purification step, the protein solution was

dialyzed against 20 mm sodium citrate buffer (pH 6.5), with

1 mm benzamidinium chloride The dialyzed protein was

divided into 0.5 mg aliquots, lyophilized, and stored at

)20 C

Crystallization

Before crystallization, protein samples were dissolved in

150 mm sodium phosphate buffer (pH 7.5), passed through

a 0.22 lm Millipore filter, and subjected to size exclusion

chromatography using a GE Healthcare HiLoad 16⁄ 60

Superdex 200 prep grade column Fractions containing

monomeric HCC-stab1 were combined and concentrated

on Microcon filters (10 kDa cut-off) to 10 mgÆmL)1 The

final buffer concentration was < 20 mm

Crystallization experiments were performed at 19C,

using the hanging-drop vapor-diffusion method and a grid

screen with different concentrations of ammonium

phos-phate versus pH Drops were mixed from 1 : 1 (v⁄ v) ratios

of the protein and precipitant solutions The best crystals

(0.35· 0.26 · 0.26 mm) grew above a reservoir containing

1.25 m ammonium phosphate (pH 7.0)

Data collection and processing

A preliminary X-ray diffraction dataset extending to 2.6 A˚

resolution (data not shown) was collected at beamline I911-3

in MAX-lab (Lund, Sweden) Later, a new dataset extending

to 1.7 A˚ resolution was collected at beamline BL14.1 of

the BESSY synchrotron (Berlin, Germany), using a

Rayonics MX-225 3· 3 CCD detector Crystals were

cryo-protected in mother liquor supplemented with 25% (v⁄ v)

glycerol, and flash-vitrified at 100 K in a nitrogen gas

stream All diffraction data were processed and scaled in the

Laue class 6⁄ m with hkl2000 [49] A summary of data

collection and processing is presented in Table 1

Structure solution and refinement

The structure was solved by molecular replacement with chicken cystatin (PDB code: 1CEW) as a search probe, using phaser [50] Manual model building was performed

in coot [51], and crystallographic refinement was per-formed with refmac5 [37] Despite successful phasing with molecular replacement in space group P61, the refinement had high R and Rfree values of 0.314 and 0.394, respec-tively Re-examination of the space group assignment and

of the diffraction data for the possibility of crystal twin-ning, carried out in the ccp4 programs truncate and detwin[52], as well as in cns [53], gave a strong indication

of hemihedral twinning, with a twin operation (h, – h – k, – l) corresponding to the higher Laue class symmetry

6⁄ mmm The same conclusions were reached for the second dataset, which was used for all subsequent calculations The final refinement was carried in refmac5, with the tls [54] and twin options included The refinement con-verged with a final R-factor of 0.138 (Rfree= 0.167) for all data, and a model characterized by rmsd from ideal bond lengths of 0.019 A˚, with 93.7% of all resi-dues in the most favored areas of the Ramachandran plot (no residues in disallowed regions) The refine-ment statistics are shown in Table 1

Acknowledgements

This work was supported, in part, by grants from the Polish State Committee for Scientific Research (Project

no 4 T09A 039 25) and from the Swedish Science Research Council (Project no 5196), by a subsidy from the Foundation for Polish Science to M Jaskolski, and by a postdoctoral fellowship (Project no PBZ⁄ MEiN⁄ 01 ⁄ 2006 ⁄ 06) from the Polish Ministry of Science and Higher Education to R Kolodziejczyk Some of the calculations were performed in the Poznan Metropoli-tan Supercomputing and Networking Center

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