structures of human proteinase 3 and neutrophil elastase – so similar yet so different

REVIEW ARTICLE

Structures of human proteinase 3 and neutrophilelastase – so similar yet so differentEric Hajjar1, Torben Broemstrup2,3, Chahrazade Kantari4, Veronique Witko-Sarsat4,5

and Nathalie Reuter3,6

1 Dipartimento di Fisica, University of Cagliari (CA), Italy

2 Department of Informatics, University of Bergen, Norway

3 Computational Biology Unit, BCCS, University of Bergen, Norway

4 Inserm, U845 and U1016, Paris, France

5 Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), France

6 Department of Molecular Biology, University of Bergen, Norway

Introduction

Human neutrophil elastase (hNE), human cathepsin G

(hCatG) and human proteinase 3 (hPR3) (also termed

myeloblastin [1] and p29b [2]) are serine proteases

mostly expressed in polymorphonuclear neutrophils,

but also are found in monocytes. All three enzymes

are homologous, although hNE and hPR3 share 56%

Keywords

human neutrophil elastase; inflammation;

myeloblastin; neutrophil, proteinase 3;

vasculitis; Wegener granulomatosis

Correspondence

N. Reuter, Department of Molecular

Biology, University of Bergen,

Thormohlensgt 55, N-5008 Bergen, Norway

Fax: +47 555 84295

Tel: +47 555 84040

E-mail: [email protected]

(Received 22 January 2010, revised 11

March 2010, accepted 18 March 2010)

doi:10.1111/j.1742-4658.2010.07659.x

Proteinase 3 and neutrophil elastase are serine proteinases of the polymor-

phonuclear neutrophils, which are considered to have both similar localiza-

tion and ligand specificity because of their high sequence similarity.

However, recent studies indicate that they might have different and yet

complementary physiologic roles. Specifically, proteinase 3 has intracellular

specific protein substrates resulting in its involvement in the regulation of

intracellular functions such as proliferation or apoptosis. It behaves as a

peripheral membrane protein and its membrane expression is a risk factor

in chronic inflammatory diseases. Moreover, in contrast to human neutro-

phil elastase, proteinase 3 is the preferred target antigen in Wegener’s gran-

ulomatosis, a particular type of vasculitis. We review the structural basis

for the different ligand specificities and membrane binding mechanisms of

both enzymes, as well as the putative anti-neutrophil cytoplasm autoanti-

body epitopes on human neutrophil elastase 3. We also address the differ-

ences existing between murine and human enzymes, and their consequences

with respect to the development of animal models for the study of human

proteinase 3-related pathologies. By integrating the functional and the

structural data, we assemble many pieces of a complicated puzzle to pro-

vide a new perspective on the structure–function relationship of human

proteinase 3 and its interaction with membrane, partner proteins or cleav-

able substrates. Hence, precise and meticulous structural studies are essen-

tial tools for the rational design of specific proteinase 3 substrates or

competitive ligands that modulate its activities.

Abbreviations

a1-PI, a1-proteinase inhibitor; ANCA, anti-neutrophil cytoplasm autoantibody; hNE, human neutrophil elastase; hPR3, human proteinase 3;

human cathepsin G, hCatG; mbPR3, membrane hPR3; PR3, proteinase 3.

2238 FEBS Journal 277 (2010) 2238–2254 ª 2010 The Authors Journal compilation ª 2010 FEBS

sequence identity for the mature enzymes, whereas

their similarity to hCatG is approximately 35%. Neu-

trophil serine proteinases are considered to be crucial

elements in neutrophil effector mechanisms [3,4]. Using

knockout mice invalidated for either hNE or hCatG, it

has been clearly demonstrated that both enzymes are

required for complete and adequate microbicidal activ-

ity [5,6]. Despite the lack of hPR3 knockout mice, a

similar function has been assigned for PR3 [2]. In

addition, it is now clear that these enzymes are also

involved in non-infectious inflammatory processes and

cell signaling [7,8]. A salient feature of hPR3 is its

identification as the main target antigen of the anti-

neutrophil cytoplasm autoantibodies (ANCA) in a

particular type of vasculitis, the Wegener’s granuloma-

tosis, which is a systemic inflammatory disease involv-

ing the lung, skin and kidney. The mechanisms

underlying this specific autoimmunization against

hPR3, and not against its homologs such as hNE, are

still unknown [9]. Because of the high sequence similar-

ity between hNE and hPR3, the substrate specificity

and the resulting functions of hPR3 have often been

extrapolated from the available data on hNE.

Together with the lack of structural and biophysical

studies on hPR3, relative to hNE, this has contributed

to a functional annotation of hPR3 that is too simplis-

tic. In recent years, however, more attention has been

paid to the structural properties of PR3 [10–14]. A bet-

ter understanding of the structure–function relation-

ship of the enzyme will contribute to the elucidation of

its original functions and help with the design of spe-

cific substrates for one or the other enzyme. So far, the

identification of the presence and hence the respective

role of either hPR3 or hNE in complex biological

models (both in vitro and in vivo) is impaired by a lack

of reliable specific inhibitors.

We review the sequence and structural data available

on both enzymes and highlight their similarities and

differences. We then summarize and discuss the latest

findings on three particular aspects of hPR3: ligand

specificity, membrane binding and putative ANCA

epitopes. We also address the similarities and differ-

ences between murine and human enzymes, and their

consequences with respect to the development of

animal models for the study of human PR3-related

pathologies.

PR3 and NE are highly similarchymotrypsin-like serine proteases

All serine proteases are named after the nucleophilic

serine in their active site. The family of serine prote-

ases comprises four distinct clans, named after

proteins representative of each clan: chymotrypsin,

subtilisin, carboxypeptidase Y and caseinolytic prote-

ase [15]. PR3 and NE are chymotrypsin-like serine

proteases. Despite the absence of any conservation of

secondary or tertiary structure elements, the four

clans of serine proteases all have the same active site

consisting of three amino acids: His, Asp and Ser.

The relative orientation of the histidine, serine and

aspartic acid is similar in all clans and results in the

formation of strong hydrogen bonds between histi-

dine and serine on the one hand, and histidine and

aspartic acid on the other hand. By convention, in

chymotrypsin-like serine proteases, the histidine,

aspartic acid and serine are numbered 57, 102 and

195, respectively. The reaction mechanism is illus-

trated in Fig. 1A. The substrate is positioned opti-

mally in the active site as a result of a network of

interactions that extends on both sides of the cleav-

able bonds. The interactions sites are named using

the Schechter and Berger nomenclature [16]. The rec-

ognition or subsites of the enzyme are Sn, … S1,

S1¢, … Sn¢ and, for the corresponding peptide,

Pn, … P1, P1¢, … Pn¢, where P1-P1¢ is the cleavable

peptide bond (Fig. 1B).

The sequences of the hNE (EC 3.4.21.37) [17–20] and

hPR3 (EC 3.4.21.76) [21–23] are shown in Fig. 2A,

where we use bovine chymotrypsinogen A numbering.

This numbering convention is used throughout the

present review (for correspondence with other number-

ing schemes, see Table S1).

hPR3 and hNE are synthesized as inactive zymogens

of 256 and 267 amino acids, respectively. These pre-

proforms undergo four consecutive steps that lead to

the mature enzymes [24–28]. The signal peptides (27

and 29 amino acids for hPR3 and hNE, respectively;

light blue boxes in Fig. 2A) are removed to yield the

proforms. The hPR3 proform is then glycosylated on

amino acids Asn113 and Asn159, and hNE on Asn

109 and Asn159 (green stars in Fig. 2A). Subse-

quently, the N-terminal dipeptide (AE for hPR3, SE

for hNE; light green boxes in Fig. 2A) is cleaved by a

cysteine protease, cathepsin C. The cleavage of the

dipeptide leads to a structural rearrangement of the

N-terminal region, which, from an extended solvent-

exposed conformation, becomes inserted into the pro-

tein core and interacts with Asp194. The enzymes then

become catalytically active. The fourth step is the

cleavage of the C-terminal pro-peptides (orange boxes

in Fig. 2A).

hPR3 and hNE are homologous and their mature

forms, comprising 221 and 218 amino acids, respec-

tively, share 56% sequence identity. Conserved resi-

dues are spread rather equally along the sequences.

E. Hajjar et al. Structure–function relationship of PR3 versus NE

FEBS Journal 277 (2010) 2238–2254 ª 2010 The Authors Journal compilation ª 2010 FEBS 2239

Fold and surface properties

X-ray data

Like all chymotrypsin-like serine proteases, hPR3 and

hNE adopt a fold consisting of two b-barrels made

each of six anti-parallel b-sheets (Fig. 2B). The struc-

tures of the mature forms of the human enzymes were

revealed by X-ray crystallography in the late 1980s for

hNE, and some years later for hPR3. To date, there

are seven structures of hNE deposited in the Protein

Data Bank (PDB code: 1B0F [29], 1H1B [30], 1HNE

[31], 1PPF [32], 1PPG [33], 2RG3 [34] and 2Z7F [35])

and one of hPR3 (1FUJ [36]). No structures of NE or

PR3 from other species are available (only computa-

tional models of the murine species have been

described) [11,14], nor are structures of the proforms.

hNE crystals all contain monomers (1B0F, 1HNE,

1PPF, 1PPG, 2RG3 and 2Z7F) or dimers (1H1B),

whereas hPR3 was crystallized as a tetramer, which

can be regarded as a dimer of dimers; two monomers

in a dimer are oriented so that their active sites face

each other, preventing the binding of large substrates.

Moreover, the hole in the middle of the tetramer is

lined with hydrophobic residues. This characteristic of

the crystals of hPR3 has not been observed in the case

of hNE, although it might be related to an increased

propensity of monomeric hPR3 to interact with hydro-

phobic environments through this region. Both hPR3

and hNE contain the same four disulfide bridges

between cysteine pairs 42–58, 136–201, 168–182 and

191–220.

The overall structural differences between hNE and

hPR3 are very small. The rmsd, calculated after struc-

tural alignment with stamp [37], on C-alpha atoms of

hPR3 and all seven available hNE structures is below

1 A and the structural difference between the seven

hNE structures is in the range 0.3–0.6 A. The loop

between extended sheets 1 and 2 (amino acids 36–39)

is the only region showing a significantly larger struc-

tural variation as a result of the insertion of three resi-

dues (NPG) in hPR3 (Fig. 2A).

Different glycosylation sites

Most structures of hNE (1B0F, 1H1B, 1PPF, 1PPG

and 2Z7F) reveal the presence of two sugar moieties

on both Asn109 and Asn159, whereas one of the latest

A

BP1′ P2′

Fig. 1. (A) Reaction mechanism of serine

proteases. (1) The first step of the catalytic

reaction, after the formation of the enzyme–

substrate or Michaelis complex, is the acyla-

tion step; it starts with the attack of the

catalytic serine on the carbonyl group of the

cleavable amide bond and the transfer of

the hydroxyl hydrogen of the serine to the

histidine. (2) This leads to the release of the

C-terminal end of the substrate and the

formation of a covalent intermediate (i.e. the

acyl enzyme) between the enzyme and the

N-terminal part of the substrate. (3) The

second step of the reaction (termed deacyl-

ation) starts with the attack of a nucleophilic

water on the substrate carbonyl and

(4) ends with the release of the N-terminal

part of the substrate, when the catalytic

triad is regenerated. The nitrogen atoms of

residues Gly193 and Ser195 stabilize the

so-called oxyanion hole. (B) Schechter and

Berger convention for the numbering of

enzyme-ligand binding sites.

Structure–function relationship of PR3 versus NE E. Hajjar et al.


structures shows only one occupied site (Asn159 for

2RG3). In the structure of hPR3, only one glycolysa-

tion site (Asn159 and not Asn113) is occupied by a

sugar. All three glycosylation sites (Asn109, Asn113

and Asn159) are remote from the catalytic triad and

the ligand binding sites (Figs 3 and 4A). According to

Specks et al. [38], both sites are occupied in neutrophil

hPR3, although they have different functional signifi-

cance; glycosylation on Asn159 influences hPR3

thermostability and increases significantly the catalytic

activity measured on a small peptidic substrate

(N-methoxysuccinyl-Ala-Ala-Pro-Val), whereas glyco-

sylation on Asn113 appears to be important (but

is not an absolute requirement) for an efficient

N-terminal processing of hPR3. Interestingly, of both

glycosylation sites, Asn113 is the furthest away from

the N-terminal end of hPR3, as observed from the

structure of the mature form. Unfortunately, no

A

B

Fig. 2. Sequence alignment and superimpo-

sition of the 3D structures of hNE and hPr3.

(A) Sequence alignment. The numbering

follows the chymotrypsin convention. Amino

acids present in the proforms are

highlighted with boxes of different colors:

signal peptides, blue; N-terminal dipeptides,

green; C-terminal propeptides, orange.

Green stars are used to highlight the amino

acids of the catalytic triad (His57, Asp102,

Ser195), whereas orange stars highlight the

glycosylation sites. The secondary structure

elements are conserved in both proteins

and are represented below the sequences

(pink arrows, extended strands; yellow

cylinders, helices). The extended strands

constituting the b-barrels are numbered 1–6

and 7–12 for the first and second barrels,

respectively. (B) Superimposition of the 3D

structures of hPr3 (1FUJ) [36] and hNE

(1PPF) [32]. Secondary structure elements

are colored as shown in Fig. 1. The catalytic

triad is represented in green balls and

sticks. Two cylinders (black lines) represent

the limits of the two b-barrels.



structure of any of the proforms of hPR3 or hNE is

available, although X-ray structures of proforms of

homologues have been reported (pro-granzyme K,

1MZA [39], chymotrypsinogen, 2CGA [40], and tryp-

sinogen, 2TGT [41]), where it can be clearly seen that

the N-terminal end (residues 14–17) freely extends out

of the core of the protein along the extended sheet

containing residue 159 (extended sheet numbered 8

in Fig. 3). Residue 159 in chymotrypsinogen and

pro-granzyme K is thus very close to the extended

N-terminal segment.

Different surface properties

Earlier studies have questioned the relationship

between the net charge difference and the functions

of PR3 and NE [36,42]. Indeed, at neutral pH, hNE

has a net charge of +10 (it contains 19 arginines and

only nine acidic residues), whereas hPR3 has a much

lower net charge of +2, although it contains approxi-

mately the same number of charged amino acids as

hNE. The sequence of the mature form contains 13

arginines, two lysines, ten aspartic acids and four

glutamic acids. Fujinaga et al. [36], as well as

subsequent in silico pKa calculations [10,43], suggest

Asp213 to be protonated. The availability of the

enzymes atomistic structures allows the calculation of

the electrostatic surface potential (i.e. the electrostatic

potential created by all the amino acids of the

enzyme in its vicinity) (Fig. 5). It is a critical determi-

nant of its surface properties and it is more relevant

to its structure–activity relationship because it reflects

not only the net charge, but also the charge distribu-

tion. Electrostatic interactions are known to play a

key role in macromolecular interactions (e.g. with

partner proteins, ions or membrane binding); thus,

they might explain protease-specific functions. Inter-

estingly, in the case of hNE, there is an omnipresence

of electropositive potential that covers most of the

surface of the enzyme, except at the substrate-binding

site. This is not the case for hPR3, where electroposi-

tive clusters (or ‘patches’) alternate with negative and

A

B

Fig. 3. Topology of hPr3 (A) and hNE (B).

Pink arrows, extended strands; yellow

cylinders, helices; green stars, catalytic

triad; orange stars, glycosylation sites; pink

circles, putative membrane binding site;

blue triangles, amino acids involved directly

in ligand binding. The extended strands

constituting the b-barrels are numbered 1–6

and 7–12 for the first and second barrels,

respectively.



neutral patches. This pattern has been shown to be

particularly suited for peripheral membrane binding

of other proteins [44–46]. Moreover, the electrostatic

surface properties of hPR3 and hNE around the

active site plays an important role in the substrate

specificity of the enzymes [10,11] and in their propen-

sity to be part of multiprotein complexes.

Substrate specificity differencebetween human NE and PR3

Kinetic studies on hNE and hPR3

Many studies have been devoted to the understanding of

hNE specificity [47–54]. Substrates with a hydrophobic

side chain at P1 are efficiently cleaved by hNE and

A

B

Fig. 4. Localization of important functional amino acids on the 3D

structure of hPR3. (A) Catalytic triad (green balls and sticks), disul-

fide bridges (yellow sticks) and glycosylation sites (orange van der

Waals spheres). (B) Amino acids directly involved in ligand binding

on hPR3 are represented by balls and sticks colored by atom type

(red, oxygen; dark blue, nitrogen; blue, carbon). The putative mem-

brane binding site [13] (or NB1 binding site) [106] is represented by

balls and sticks colored magenta (hydrophobic amino acids: F165,

F166, L223 and F224) and pink (acidic amino acids: R177, R186A,

R186B, K187 and R222).

A

B

Fig. 5. Calculated electrostatic potential of hPR3 (A) and hNE (B).

The electrostatic potential is mapped on the molecular surface of

the enzyme and colored in blue (+5 kT), white (0 kT) and red

()5 kT). The equipotential contours are also represented at +1 kT

(transparent blue) and )1 kT (transparent red).



P1-valine is preferred over an alanine or a phenylala-

nine. Substrates with P1-Ile can also be hydrolyzed

[48] by hNE. Peptides with P1-Met residues can also

be cleaved, whereas oxidation of the methionine

decreases the binding to hNE [50]. Extension of the

peptide chain results in significant increases in catalytic

efficiency and P1-specificity becomes broader with

decreasing chain length. Compared to hNE and other

serine proteases, there are relatively few available

results on the specificity of PR3. The earliest studies

revealed the preference of PR3 for ‘small hydrophobic

amino acids’ such as alanine, serine and valine [55].

Brubaker et al. [56] emphasized that norvaline is pre-

ferred to valine, which itself is better than an alanine.

Substrates with a methionine at P1 would also be effi-

ciently cleaved by PR3 [57,58]. These studies focused

on the P1 amino acid and described differences mostly

resulting from the size or volume of the S1 pocket.

Most importantly, they could not rely on structural

data because no structure of PR3 was then available.

Regarding hNE, most studies used a substrate with

chromophoric leaving groups (i.e. these do not extend

with amino acids in P¢ sites), which explains why the

importance of the P¢–S¢ interaction has been over-

looked for many years. Recently, however, the

importance of S¢–P¢ interactions has been clearly

established for hPR3 [54,58]; substrates extending

beyond P1¢ have a systemic favorable effect on PR3

catalysis.

Insights from the abundant structural X-ray data

on hNE

The first NE structure solved in 1986 was co-crystal-

lized with the third domain of the turkey ovomucoid

inhibitor (OMTKY3) (1PPF) [32], which is a canonical

protein inhibitor of the Kazal family. It is bound

noncovalently and provides structural insights into

how peptidic substrates bind to NE in the Michaelis

complex. Recently, the crystal structure of hNE with

another proteic inhibitor was released (2Z7F) [35]; the

secretory leukocyte protease inhibitor is a secreted

inhibitor that protects epithelial tissues from serine

proteases. Additional NE structures were solved with

synthetic inhibitors in the binding site, including chlo-

romethyl ketone peptides MeO-Suc-Ala-Ala-Pro-Val

(1PPG) [33] and MeO-Suc-Ala-Ala-Pro-Ala (1HNE)

[31] and an orally active peptidyl pentafluoroethyl

ketone (1B0F) [29]. The structure of hNE with low-

molecular weight inhibitors has also been reported;

MacDonald et al. [30] reported the structure of NE

with a nonpeptidic inhibitor, a pyrrolidine trans-

lactame which also binds covalently to Ser195 (1H1B).

Later Huang et al. [34] reported the structure of NE

with another mechanism based (suicide) inhibitor, a

derivative of a 1,2,5-thiadiazolidin-3-one 1,1-dioxide

(2RG3). This scaffold has been described as particu-

larly suited for the design of specific NE-inhibitors

[59]. Mechanism-based inhibitors are covalently linked

to the active site (either Ser195 or both Ser195 and

His57) and mimic either the conformation of a tetrahe-

dral intermediate (tetrahedral adduct) or of the acyl

enzyme.

Thus, unlike hPR3, hNE has been crystallized with

several ligands (see list in Table 1); these structures

provide information about the interactions possible

between the enzyme and different types of substrates,

and thereby about its specificity much more extensively

than in the case of hPR3. The different X-ray struc-

tures show that pockets S1–S4 are mostly made of

hydrophobic amino acids [29,30,32,33], with the excep-

tion of the side chain of Arg217, which can stabilize

polar or acidic groups at P4 [32] and even at P5 [33].

Interestingly, the benzyl of the N-protecting group in

Table 1. X-ray structures of neutrophil elastase and proteinase 3 available in the Protein Data Bank (PDB). The names and types of inhibitors

present in the active site of the enzymes are listed, as well as the type of complex that they make with the structure, characterized by the

reaction intermediate that they resemble the most. Resolution is given in angstroms.

Enzyme Substrate name or formula Substrate type

PDB code and

resolution

hNE OMTKY3a Protein (Kazal family) Michaelis complex-like 1PPF [32] 1.80

Secretory leukocyte protease inhibitor (SLPI)b Protein (chelonianin family) Michaelis complex-like 2Z7F [35] 1.70 [34]

MeO-Suc-Ala-Ala-Pro-Val-CH2Cl Peptidyl chloromethyl ketone Tetrahedral adduct 1PPG [33] 2.30

MeO-Suc-Ala-Ala-Pro-Ala-CH2Cl Peptidyl chloromethyl ketone Tetrahedral adduct 1HNE [31] 1.84

N-(4-(4-morpholinylcarbonyl)benzoyl)-Val-Pro-Ile-C2F5 Peptidyl pentafluoroethyl ketone tetrahedral adduct 1B0F [29] 3.00

GW475151 Pyrrolidine trans-lactame Acyl enzyme-like 1H1B [30] 2.00

4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid 1,2,5-Thiadiazolidin-3-one 1,1-dioxide Acyl enzyme-like 2RG3 [34] 1.80

hPr3 None – 1FUJ [36] 2.20

a Turkey ovomucoid third domain. b Secretory leukocyte protease inhibitor.



1B0F forms a p–p stacking interaction with Phe215.

An additional feature of the ‘unprimed subsites’ is that

residues 214–216 form an antiparallel b-sheet with

backbone atoms of residues P1–P3 of protein and

peptidyl inhibitors which is known as the ‘canonical

conformation’ [60]. The primed substrate binding sites

are less well defined; the low molecular weight com-

pounds do not extend far into the prime sites, and the

same applies for the peptidyl inhibitors. Proteic inhibi-

tors reveal some contacts in the prime site but, because

of their size and rigidity, it is possible that the contacts

they make with the enzymes are not exactly compara-

ble with the contacts small flexible peptides would

make. An exhaustive list of amino acids generally

described as forming the binding sites is provided else-

where [53,60].

Mapping the substrate binding sites of hPR3: the

contribution of in silico studies

The release of the first X-ray structure of hPR3 [36]

and its comparison with already known structures of

hNE has revealed some of the basis for the difference

specificity between the two enzymes, despite the lack

of a structure of a complex between hPR3 and a sub-

strate. Indeed Fujinaga et al. [36] describe the S1

pocket, but also report a list of the amino acids consti-

tuting the neighboring binding sites, which most prob-

ably form the basis for the differential substrate

specificity of hPR3 and hNE. The Ala213 to Asp213

substitution in PR3 reduces the size of the S1 pocket

(from 152.7 to 98.6 A3), making P1 more restrictive

in hPR3 than hNE. The Asp213 is observed to be

hydrogen-bonded to the carbonyl of Gly197 and its

protonation state (and increased pKa value) is proba-

bly a result of the hydrophobic environment. Indeed,

using molecular dynamics simulations and pKa calcula-

tions, we predicted a significant increase of the pKa

value of Asp213 compared to its value in solution [43].

Fujinaga et al. [36] suggest that the substitution of

Leu99 to Lys99 makes the S2 pocket of hPR3 deeper

and more polar compared to hNE, and that it

increases the polarity in S4. The substitutions of

Arg217 to Ile217 and Gly218 to Trp218 in hPR3

compared to hNE should render the S5 binding site

more hydrophobic. For the primed subsites of hPR3,

the most significant substitution is Asn61 to Asp61.

Consequently, hPR3 should prefer basic residues at

P1¢ and P3¢. The substitution of Ile151 to Pro151 and

Ile143 to Arg143 will create a basic S2¢ site favoring

the binding of acidic residues.

However, because there is no X-ray structure of

hPR3 with a substrate in its active site, there is no

accurate description of the ligand–enzyme interactions

such as there is for hNE. In an attempt to fill this gap,

we performed molecular dynamics simulations of

hPR3 and hNE complexed with peptides of varying

sequences and sizes [10–12]. Indeed, molecular dynam-

ics simulations are very well suited and widely used

[61–63] for providing both structural and dynamical

information, and thus neatly complement experimental

techniques such as X-ray and NMR. These simulations

have provided structures at the atomic level of detail

of hPR3, a map of the S and S¢ sites (Fig. 6) and a

description of the interaction scheme for six different

peptidic ligands. The simulations show no direct inter-

action between Asp213 and the P1 amino acids,

suggesting that a polar amino acid in S1 is not

something that hPR3 can accommodate better than

hNE. Relatedly, Asp213 does not appear to have a

role in specificity, in agreement with that suggested by

Fujinaga et al. [36]. The specificity difference between

the two enzymes lies in S2, S1¢, S2¢ and S3¢, which are

all more polar in hPR3 than in hNE. S2 in hPR3 is

clearly suited to accommodate a negatively-charged

amino acid, such as Asp, with Lys99 and Arg60 acting

as potential hydrogen bond donors. It is interesting to

note that Lys99 contributes to both the hydrophobic

S4 site and to the polar S2 site, playing two different

roles. Similarly S1¢ and S3¢ are inter-connected sites of

polar character because they are made of Asp61 (as

A

B

Fig. 6. Map of the recognition sites of hPr3 (A) and hNE (B). Inven-

tory of the principal amino acids located in the recognition subsites

of the hPR3 (A) and hNE (B) and interacting directly with the sub-

strates [10,11] (only residues interacting with side chains of the

peptide are listed).



well as other amino acids). It participates in the two

interaction sites and interacts with lysine or arginine

side chains of the ligand. S2¢ is characterized by

Arg143, which can interact through hydrogen bonds

with a negatively-charged amino acid at the P2¢position of the substrate sequence. Ile190 of hPR3 is

not observed to be in close contact with the P1 side

chain but Val216 constitutes the main interaction part-

ner in the S1 site, together with the CbH2 group of

Ser195. The S4 and S3 sites are hydrophobic in both

hPR3 and hNE. Another important conclusion derived

from the molecular dynamics simulations is that the

seven sites are shown to be interconnected; in particu-

lar, S4 and S2 share Lys99, which plays both a hydro-

philic and a hydrophobic role, with the latter being

achieved as a result of the CH2 groups of its long side-

chain. S1¢ and S3¢ overlap and share Asp61, and the

lack of a P¢–S¢ interaction modifies the network of S–P

interactions. This is an important result in the context

of drug design and the investigation of enzyme–ligand

binding sites by directed mutagenesis experiments. At

least as important is the finding that different amino

acids of the enzyme can alternatively participate in one

given recognition site or another depending on the nat-

ure of the sequence of the ligand. This illustrates the

adaptability of the enzyme; most of the recognition

sites are not rigid predefined pockets. Rather, they are

regions at the surface of the enzyme that have a signifi-

cant degree of flexibility and can adapt to the

substrate.

Table 2 summarizes the preferred amino acid types

at each P or P¢ site and it can be used to design pep-

tidic substrates specific of one enzyme or the other.

Using these data, we have designed the peptide

sequence VADVKDR and demonstrated that it is

highly specific for hPR3 versus hNE [10]. We find that

the cleavage site of p21 ⁄waf1 (QEA-RER) [64] also

conforms to the pattern listed in Table 2. Knowledge

of the sequence pattern binding to hPR3 can also be

used to help identify novel endogenous substrates of

the proteinase.

PR3 and NE interact differently withthe PMN membrane

Divergence of hypotheses from experimental

studies

Several studies have attempted to characterize the

association of PR3 and NE with the neutrophil mem-

brane. It has been shown that only PR3, and not NE,

is already present at the plasma membrane of inacti-

vated PMN [65,66]. We have previously reported a

specific association of PR3 with the plasma membrane,

which is stronger than simply an ionic interaction [67],

and some studies argue in favor of a weak charge-

dependent mechanism similar for the two proteases

[42].

A number of proteins have been experimentally

identified as potential partners of PR3 at the mem-

brane, which might be of critical importance for its

functions, as well as for our understanding of its

involvement in Wegener’s granulomatosis [68,69].

Aviram and colleagues provided evidence of colocal-

ization of PR3 with the integrin CD11b ⁄CD18 (b2

integrin) [70], the Fcgamma receptor FcgRIIIb and the

p22phox subunit of cytochrome b558 in the membrane,

and PR3 is demonstrated to be localized in lipid raft

domains [71,72]. More recently, Bauer et al. [73]

reported the expression of membrane hPR3 (mbPR3)

and CD177 (NB1) on the same subset of neutrophils,

whereas von Vietinghoff et al. [74] proposed that PR3

membrane expression could be mediated through

CD177 binding. Interestingly, we demonstrated that

PR3 could be externalized at the plasma membrane

during the very early stages of apoptosis in the absence

of degranulation, thus strongly suggesting an extra-

granular pool of PR3 [75]. Most interestingly, we

reported a co-localization of PR3 with phospholipid

scramblase 1, a myristoylated membrane protein pres-

ent in lipid rafts and involved in the redistribution of

membrane lipids during neutrophil apoptosis [76].

Co-immunoprecipitation studies performed on neutro-

phil lysates provided evidence indicating that both

PR3 and phospholipid scramblase 1 were associated

within the same protein complex. However, to our

knowledge, there is no evidence of a physical interac-

tion between mbPR3 and any of these potential

partners, highlighting the lack of biophysical and

structural data on the membrane binding mechanism

of PR3. It should be noted that the diversity of the

potential PR3 partners might reflect specific functions

and especially regulatory functions in neutrophil

activation or apoptosis, which are currently being

unravelled.

Table 2. Inventory of the amino acids types preferred at sites P6–

P4¢ (h, hydrophobic; ), acidic; +, basic; p, polar) of the human and

mouse PR3 and NE [10,11].

P6 P5 P4 P3 P2 P1 P1¢ P2¢ P3¢ P4¢

hPR3 h h ) h ⁄ p + ) +

hNE h h h h h ⁄ p h

mPR3 + ) h h h ⁄ + h ⁄ p + ) +

mNE h ) h h h ⁄ p ) ) ) )



By contrast, a direct interaction between hPR3 and

lipid vesicles has been demonstrated by Goldman et al.

[77]. They investigated the interaction of hPR3, hNE

and human myeloperoxidase (an enzyme also present

in neutrophil azurophilic granules), with reconstituted

lipid bilayers containing zwitterionic and anionic

phospholipids; pure dimyristoylphosphatidylcholine,

dimyristoylphosphatidylcholine ⁄dimyristoylphosphat-

idylglycerol (70 : 30, 50 : 50 or 30 : 70) and pure

dimyristoylphosphatidylglycerol. Their results show

that the molar affinity of hPR3 is better than the one

of hNE and myeloperoxidase for all mixtures with

anionic lipids, and that only hPR3 anchors to neutral

bilayers. According to their findings, hPR3 associates

with the mixed bilayer (50 : 50) with strong hydropho-

bic interactions (i.e. by inserting hydrophobic amino

acids in the lipid bilayer), whereas hNE and myelop-

eroxidase only have weak hydrophobic interactions

with the lipids. Interestingly, they report that bilayer-

bound hPR3 has a reduced catalytic efficiency,

whereas its inhibition by a1-antitrypsin is more impor-

tant than that observed for the soluble form of the

enzyme. The latter is in contradiction to other studies

[42,78]. It has been shown that hPR3 enzymatic activ-

ity is not inhibited by its inhibitors a1-proteinase

inhibitor (a1-PI, 52 kDa) and elafin (6 kDa) when

bound to the outer cell surface of neutrophils, but

only by a low-molecular-weight protease inhibitor

(phenylmethansulfonyl fluoride). This suggests that

a1-PI and elafin are not active on mbPR3 as a result

of steric hindrance.

On the sole basis of visual inspection of the 3D

structure, Goldman et al. [77] suggest that the region

constituted by Phe166, Ile217, Trp218, Leu223 and

Phe224 might be involved in the insertion of hPR3

into the lipids. This study has shown that hPR3 can

bind to lipid bilayers directly (i.e. with direct physical

interaction between amino acids of the protein and

lipids) and is stable without any other partner. It

strongly suggests that it can bind with the same

mechanism to cellular membranes.

Structural determinants predicted by in silico

studies

Although the application of NMR and X-ray meth-

ods to peripheral membrane proteins remains chal-

lenging, theoretical approaches have proven extremely

valuable and reliable for studying their structure and

membrane-binding mechanisms [79–85]. We used

molecular dynamics simulations with implicit mem-

brane representation to allow for a cost-effective

investigation of the binding of PR3 and NE to the

lipid bilayers, in the presence of different types of

lipid bilayers that vary with respect to their anionic

character. The results obtained show that, unlike

hNE that binds only to negatively-charged mem-

branes, hPR3 is able to bind both anionic and neu-

tral membranes but with a preference for negatively

charged lipids; the calculated binding energies are

)10.87 and )3.07 kcalÆmol)1 for anionic and neutral

membranes, respectively. Furthermore, the simulations

of the binding mechanism reveal a unique membrane-

binding site and binding mechanism of PR3. It

involves a few basic amino acids that orient PR3

towards the membrane in the correct direction to

allow it to insert a hydrophobic patch comprising

F165, F166, F224, L223, F184 and W218. These resi-

dues are carried by surface loops (Figs 3A and 4B)

and in silico mutations abolish membrane association.

NE follows a different binding mechanism, where dif-

ferent regions of its highly electropositive surface

(Fig. 5B) are able to interact with anionic lipids, but

without insertion of hydrophobic amino acids, and

thereby in a much shallower way than PR3.

A mutagenesis analysis performed in rat basophil

leukaemia (RBL) cells transfected with wild-type PR3

or PR3 mutated within the hydrophobic patch demon-

strated that the latter was essential for membrane

insertion [69]. The mechanism of membrane anchoring

described by Goldman et al. [77] and ourselves (i.e.

electrostatically driven attraction by basic amino acids

and insertion of hydrophobic amino acids directly in

the membrane) is not incompatible with the presence

of a partner protein such as NB1. Such interactions

might simply reinforce or perpetuate the membrane

anchorage and ⁄or facilitate the function(s) of mem-

brane-bound PR3.

The quest for PR3-ANCAs epitopes

PR3 is the main target autoantigen of ANCAs, which

predominate in patients with Wegener’s granulomato-

sis. There is growing evidence that ANCAs have a

pathogenic role in systemic vasculitis [86–88]. Although

some controversy remains about the mechanisms of

this pathogenicity [89], the most plausible explanation

involved binding to (and activation of) neutrophils by

ANCAs [90]. The binding of ANCAs to neutrophils is

only made possible by the availability of PR3 at the

PMN surface; in other words, by the physical associa-

tion of PR3 with the PMN membrane. The identifica-

tion of disease-inducing epitopes of hPR3 will

constitute a critical step toward the development of

epitope-specific therapeutic strategies. However, the

quest for the epitopes of ANCAs on hPR3 has turned



out to be a challenging task. Many attempts have been

made to characterize the ANCAs epitopes on PR3

[91–98]. It is now clear that ANCAs recognize several

regions of PR3. Even though it has been shown that

they recognize conformational epitopes [91] and that

they do not bind the denatured enzyme, many studies

erroneously used linear peptides to probe ANCAs epi-

topes of PR3 [92,95,99]. There is little overlap between

the results obtained in these studies (Fig. 7A–C).

Moreover, although the epitopes suggested by Wil-

liams (Fig. 7A) are all located in surface-exposed loops

of the structure, some of the regions suggested by Grif-

fith et al. [99] and van der Geld et al. [95] include sec-

ondary structure elements of hPR3 (Fig. 7B,C). The

amino acids belonging to putative epitopes commonly

suggested by the three studies are represented by green

van der Waals balls in Fig. 3D: (143)RV(144),

(178)PHN(180) and (186)PRRKAGIC(191). The latter

two overlap with or are very close to the predicted

membrane binding site. In accordance with the

hypothesis that steric hindrance will prevent ANCA

from binding to regions of PR3 too close to the mem-

brane, one can rule out these putative epitopes

(178)PHN(180) and (186)PRRKAGIC(191). The same

reasoning can be used to assess or rule out epitopes

suggested by future studies.

Murine enzymes

Substrate specificity differs from the human

enzymes

Specks and coworkers [100] demonstrated that the

mouse and human PR3 (mPR3 and hPR3, respec-

tively) have different physicochemical properties. Using

inhibitors and short hydrophobic peptides (not extend-

ing in the P¢ sites), they show that they have different

substrate specificities. Unfortunately, the size and the

nature of the peptides limit the conclusions that can be

drawn with respect to the P1 sites. The lack of struc-

tural information on the murine enzymes and on their

complexes with cleavable peptides makes it difficult to

A B

C D

Fig. 7. Localization of the putative linear

epitopes identified by (A) Williams et al.

[92], (B) Van Der Geld et al. [95] and (C)

Muller et al. [97] (green). (D) Amino acids

identified by the three studies (van der Wa-

als green spheres). The legend is as shown

in Fig. 2B, except that the amino acids of

the catalytic triad are colored in red.



determine the basis for their substrate specificity. We

used homology modeling to build structural models of

the murine enzymes and performed molecular dynam-

ics simulations of 14 different enzyme–peptide com-

plexes [11], and their analysis provides an inventory of

the amino acids forming the substrate binding sites of

the murine enzymes. The surface of mPR3 is globally

more electronegative than the one of hPR3 (Fig. 5),

whereas the differences between mNE and hNE are

less significant on the overall protein surfaces. In par-

ticular, substitutions of several basic amino acids

around the substrate binding sites of hPR3 change its

surface properties: R60Q, R63bQ, R74L, N98E, K99N

and G219E. Unlike hPR3, mPR3 is thus unlikely to

bind substrates with acidic groups (Asp, Glu) on the S

side. Consequently, very efficient substrates of hPR3

might be poor substrates of mPR3. The specificity dif-

ference between mPR3 and mNE lies in S1¢ and S3¢.The S2 and S2¢ sites in both enzymes might accommo-

date the same type of amino acids, although the

differences between these sites in the human enzymes

were used for designing specific substrates. Table 2

summarizes the types of amino acids preferred at sites

P6–P4¢, and also for the human and mouse enzymes.

Such an inventory will benefit the development of

highly specific substrates of each of the neutrophil

serine proteases [14].

Membrane binding site: poor sequence

conservation between species

Half of the amino acids predicted to be involved in the

membrane binding of hPR3 are not conserved in

mPR3; F166L, F184L, K187A, W218R, R222L and

L223Q. The mouse form of PR3 lacks three aromatic

and one basic amino acid compared to the human

form. The former are known, from integral membrane

proteins, to be found at the membrane interface,

whereas the latter provides electrostatic interactions

with the polar lipid heads. In addition, we have seen

that the mouse form is globally more electronegative

than the human form. As a consequence of these dif-

ferences in sequence and structural properties, mouse

PR3 is likely to be able to bind to the plasma mem-

brane using the same region as human PR3, although

less strongly and specifically, as observed by Wiesner

et al. [100].

In conclusion, molecular studies of the comparison

between human and mouse PR3 clearly point to major

differences in their catalytic properties and their ability

to interact with membranes, which is a key determi-

nant of the accessibility to a specific substrate and,

ultimately, their function.

Conclusions

It is now becoming clear that both hNE and hPR3

play an important role in cell signaling and represent

regulators of the inflammatory response. They have

been shown to be involved in a number of regulatory

pathways; however, although they are crucial to our

understanding of the inflammatory processes, many

aspects of their specific roles are still under debate

[42,101]. This review emphasizes the importance of

considering the structural characteristics of hPR3 and

hNE in studies of their pathophysiological role in gen-

eral, and, in particular, for the design of specific

ligands and ⁄or drugs. It provides a comprehensive pic-

ture of the functionally important regions of hPR3.

Interestingly, the amino acids that are responsible for

the differential substrate specificity of hPR3 compared

to hNE, and those responsible for its membrane

anchorage, lie in the C-terminal part of its sequence

(second b-barrel), which is known to be the most

important domain in shaping serine protease evolution

[102,103]. The location of these key residues in that

particular region confirms their functional significance

and provides yet another argument allowing the con-

clusion to be made that hPR3 and hNE have intrinsi-

cally different functional roles.

One key issue is the difference in the subcellular

localization between hPR3 and hNE, which might be

the consequence of a different molecular association

during targeting mechanisms that remains to be deter-

mined. This difference in subcellular localization and

its consequence with respect to serine proteinase func-

tion has been well illustrated in the cellular model of

mast cell lines transfected either with hPR3 or with

hNE. By contrast to hNE, hPR3 can cleave intracellu-

lar specific proteins involved in the regulation of intra-

cellular functions, such as proliferation and

differentiation, similar to p21 ⁄waf1 [64,104], or be

involved in apoptosis, similar to pro-caspase-3 [105].

Notably, hPR3 could cleave membrane-associated

procaspase 3 into a 22 kDa fragment, which is distinct

from the classical apoptosis-induced fragment involved

in neutrophil survival [105].

In hNE-transfected mast cell lines, and under basal

conditions, hNE subcellular localization was restricted

to granules, whereas hPR3 was localized both within

the granules and at the inner face of the plasma mem-

brane, thus allowing access to specific substrates [104].

Upon activation of degranulation, hPR3 could ulti-

mately be exposed at the cell surface. Most interest-

ingly, PR3 can be externalized during apoptosis and

impaired apoptotic cell clearance by macrophages, thus

amplifying inflammation [76]. These findings were con-



firmed in neutrophils. Although the major pool of

intracellular PR3 is within the azurophilic granules,

the extragranular pool of PR3, which is externalized

during apoptosis, might be functionally very important

in the pathophysiology of vasculitis [9]. Although

much progress has been made in recent years with

respect to our understanding of the structure–function

relationship of hPR3, the field would benefit immen-

sely from new structures of the enzyme complexed

with inhibitors, as well as from new biophysical studies

investigating its interaction with lipid bilayers and

partner proteins.

Acknowledgements

Funding for N.R. and T.B. was provided by the

National Program for Research in Functional Genom-

ics in Norway (FUGE) from the Research Council of

Norway, as well as by the Bergen Science Foundation

(Bergen Forskningsstiftelse). V.W.S. acknowledges

funding from Inserm (ANR08-GENO-035-01) and

C.K. received funds from ABCF Mucoviscidose,

‘Vaincre la mucoviscidose’ (VLM) and Societe de

Nephrologie. E.H. acknowledges the assistance of EU

Grant MRTN-CT-2005-019335 (‘Translocation’).

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Supporting information

The following supplementary material is available:

Table S1. Correspondence between the two numbering

conventions in use for mature hPR3.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

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structures of human proteinase 3 and neutrophil elastase – so similar yet so different

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