(Miten löysin tämän artikkelin tnään 23.11.2021: Olin etsimässä Sars-2 S-proteiinin trimerisoitumisesta ensinnä arikkeleita ja siten aloin pohtia transmembraanisen trimeroituneen tyven muodostumista ja etsiäö prokollageenin trimerisoivaa domeenia; - samalla löysin entsyymejä- , jotka pilkkovat tässä yhteydessä mahdollista trimeeriä koska se on transientti fysiologisesti. Tosin sars-2 ei anna trimeerien pilkoutua ainakaan ennen kuin uudet virionit ovat valmiita stabiilien trimeerien kanssa ja toisaalta infektoituminen vanhalla trimeerillä on tehty. Löysin siitä fokuksesta molekyylejä, jotka ovat Sars-2 interaktioproteiineja ja samalla rokotevalmstuksessakin tarvittava. Yhdessä oli CUB-domeeni ja NTR-domeeni vapautui siitä pilkkoutumalla. NTR-modulista sanottiin, että se oli " homologinen TIMP:n kanssa, mutta ei pystynyt vaikuttamaan metsinkiineihin". Sen takia etsin metsinkiinit esiin . Niin läysin tämän artikkelin).
https://www.jbc.org/article/S0021-9258(20)42509-8/fulltext
Catalytic Domain Architecture of Metzincin Metalloproteases
Open AccessDOI: https://doi.org/10.1074/jbc.R800069200
Metalloproteases
cleave proteins and peptides, and deregulation of their function leads
to pathology. An understanding of their structure and mechanisms of
action is necessary to the development of strategies for their
regulation. Among metallopeptidases are the metzincins, which are mostly
multidomain proteins with ∼130–260-residue globular catalytic domains
showing a common core architecture characterized by a long zinc-binding
consensus motif, HEXXHXXGXX(H/D), and a
methionine-containing Met-turn. Metzincins participate in unspecific
protein degradation such as digestion of intake proteins and tissue
development, maintenance, and remodeling, but they are also involved in
highly specific cleavage events to activate or inactivate themselves or
other (pro)enzymes and bioactive peptides.
Metzincins are subdivided
into families, and seven such families have been analyzed at the
structural level:
the astacins,
ADAMs/adamalysins/reprolysins,
serralysins,
matrix metalloproteinases (MMPs) ,
snapalysins,
leishmanolysins,
and
pappalysins.
These families are reviewed from a structural point of
view.
Metalloproteases
Cleavage
of peptide bonds is essential for life, and the factors responsible for
peptide cleavage are ubiquitous. Among them are MPs, 2
which are mostly zinc-dependent peptide-bond hydrolases. They
participate in metabolism through both extensive and unspecific protein
degradation and controlled hydrolysis of specific peptide bonds (1.).
Deregulation of such vast degrading potential leads to pathologies, and
in addition, MPs may also act as virulence factors during poisoning and
microbial infection. Such a wide range of biological functions makes
structural studies of these proteins indispensable to any understanding
of their function and to the design of novel, highly specific
therapeutic agents to modulate their activity (2.).
Most MPs are members of a protease tribe, the zincins, and they possess a short consensus amino acidsequence, HEXXH.
This motif contains two protein ligands of the catalytic zinc and a
glutamate that acts as general base/acid during the catalytic process (3.,4.).
A third metal ligand is a solvent molecule, further bound to and
polarized by the glutamate. This solvent performs nucleophilic attack on
the carbonyl carbon of the scissile bond of a bound substrate, leading
to a tetrahedral gem-diolate reaction intermediate, which is stabilized by the positively charged metal ion and neighboring protein residues (5.). Subsequent evolution of the intermediate under assistance of the general base/acid eventually leads to bond disruption.
Zincins are divided into the
gluzincin,
aspzincin, and
The latter contains mostly multidomain proteins with an N-terminal
prodomain engaged in latency maintenance, a catalytic protease domain,
and farther downstream domains engaged in protein-protein and cell-cell
interactions and other regulatory functions. The protease domain is
characterized by a C-terminally extended zinc-binding motif, HEXXHXXGXX(H/D),
with a hallmark glycine and a third zinc-binding histidine or
aspartate. In addition, a methionine is present in a conserved
downstream turn, the Met-turn (3.,7.,8.).
Metzincins split into families, seven of which have been characterized
at the structural level for at least one of their members: astacins,
ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases,
snapalysins, leishmanolysins, and pappalysins.
In addition, a series of
sequences reported from genome sequencing projects indicate there are
further structurally yet uncharacterized families, tentatively referred
to as
fragilysins,
gametolysins,
archaemetzincins,
thuringilysins,
coelilysins,
ascomycolysins,
helicolysins, and
cholerilysins (for a
detailed review, see Ref.7.).
The Metzincin Fold
To
date, >200 structures of metzincins, comprising at least the
catalytic domain, have been deposited with the Protein Data Bank (supplemental Table 1). In this review, seven lead structures of each of the aforementioned families are discussed: Astacus astacus astacin, Crotalus adamanteus adamalysin II, Pseudomonas aeruginosa aeruginolysin, human neutrophil collagenase (MMP-8), Streptomyces caespitosus neutral protease (snapalysin), Leishmania major leishmanolysin, and Methanosarcina acetivorans ulilysin (9.,10.,11.,12.,13.,14.,15.).
These prototypes cover distinct kingdoms of life and represent archaea,
bacteria, protozoa, crustaceans, reptiles, and mammals. The structures
reveal that metzincins share a common scaffold and active-site
environment, but each family has distinguishing structural elements.
Mature catalytic domains are ∼130–260-residue globular moieties that
bifurcate into an upper NSD and a lower CSD with respect to a central
active-site cleft. Substrates bind horizontally to this cleft from left
to right in an approximately extended conformation (supplemental Fig. 1 A). (All topological indications refer to the standard orientation displayed in this figure.)
The NSD displays a five-stranded twisted β-sheet at the top (except leishmanolysin, which has four strands) (supplemental Fig. 1A). All strands (βI–βV) except the fourth are parallel to each other and to any substrate that is bound in the cleft. The antiparallel strand, βIV, forms the lower edge of this subdomain and creates an upper rim or northern wall of the active-site crevice (16.). This strand binds a substrate in an antiparallel manner, mainly on its non-primed side. The loop segment connecting strands βIII and βIV (referred to as LβIIIβIV) leads to the appearance of bulge-like elements, which mainly affect subsites S1′ and S2′ (see Ref.17. for subsite nomenclature in proteases). This gives rise to extensive variations in enzyme-substrate interactions on the primed side of the active-site clefts. The NSD also contains two long α-helices, αA and αB, the backing helix and the active-site helix, respectively. Both are arranged on the concave side of the β-sheet in an identical manner in all metzincin structures (supplemental Fig. 1B). Helix αB superimposes well in all seven structures (Fig. 1A) and encompasses the first half of the zinc-binding motif, which includes the first two zinc-binding histidine residues (Fig. 1A and supplemental Fig. 1). At the end of helix αB, the polypeptide chain takes a sharp downward turn, mediated by the glycine of the consensus sequence. The main-chain angles of this residue in the different lead structures indicate that any other residue would be in a high-energy conformation and thus disfavored, with the exception of ulilysin (see below).
FIGURE 1Overlay of structural segments common to the seven prototypes as Cα plots. A,
the active-site helix αB (including the side chains of the
zinc-liganding histidines and aspartate and the general base/acid) and
the Met-turn (with the methionine side chain). Blue, astacin; cyan, adamalysin II; red, leishmanolysin; green, MMP-8; white, aeruginolysin; yellow, snapalysin; orange, ulilysin. The magenta curved arrows indicate where an extra domain is inserted in leishmanolysin. B, the upper domain β-sheet (strands βI–βV, in cyan, blue, green, orange, and yellow, respectively), the posterior helix αA (red), and the C-terminal helix αC (purple) as found in the seven leads. Leishmanolysin lacks strand βII.
The
CSD starts after this glycine, and the chain leads to the third zinc
ligand, a histidine or an aspartate, which approaches the metal from
below (supplemental
Fig. 1). This subdomain contains few repetitive secondary structure
elements, mainly a C-terminal helix αC at the end of the polypeptide
chain. Helices αB and αC are connected by structures that vary both in
length and conformation. However, all structures coincide at a conserved
1,4-β-turn containing a methionine at position 3, the Met-turn, which
is separated from the third zinc-binding histidine by connecting
segments of 6–53 amino acids in the different structures. The Met-turn
is superimposable (including the conformation of the methionine side
chain) (Fig. 1A) and is positioned underneath the catalytic Zn2+,
forming a hydrophobic pillow. However, no direct contact with the metal
is observed. Mutation studies suggested a role for this methionine in
the folding and stability of the catalytic domains, although the strict
conservation of this residue remains to be explained (18.,19.). 3
The S1′ pocket of metzincins is shaped at the top by a
protruding bulge made by LβIIIβIV and at the bottom by a wall-forming
segment made up of residues intercalated between the Met-turn and the
C-terminal helix αC (16.).
This segment diverges in structure and length (ranging from 11 to 37
residues between the Met-turn methionine and the first residue of helix
αC) in all seven of the reference structures.
The Zinc-binding Site
The catalytic zinc ion lies roughly at the center of the bottom of the active-site cleft. It is coordinated by the Nϵ2 atoms of the three consensus histidines (two histidine Nϵ2 atoms and one aspartate Oδ2
atom in snapalysin) and the catalytic solvent molecule, substituted by
other ligands in the enzyme/inhibitor-product complexes reported (supplemental
Table 1). Some metzincins display an additional protein ligand at a
slightly greater distance from the catalytic cation in the form of a
tyrosine Oη atom, as seen in unbound astacin and serralysins and as hypothesized in ulilysin (21.).
This tyrosine residue lies two positions ahead of the Met-turn
methionine. In (unbound) snapalysin, a tyrosine lies two positions
downstream of the metal-binding aspartate, though no longer within
binding distance of the zinc ion. These tyrosine residues flip back and
forth during substrate anchoring, cleavage, and product release in a
motion referred to as the tyrosine switch. In this, they may play a role
in substrate and catalytic solvent binding and stabilization of the
tetrahedral intermediate or the product amino group (
Structural Relatedness among Families
A
structure-based sequence alignment reveals that, with the exception of
the region comprising the zinc-binding consensus sequence, sequence
identity is negligible among metzincins despite evident structural
relatedness. Based on this alignment, pairwise comparisons between leads
(supplemental Fig. 2) show between 41 and 118 topologically equivalent Cα positions and root mean square deviations between 3.3 and 4.0Å, i.e. above the threshold at which two Cα positions can be considered as structurally equivalent (3.0 Å) (
25.). The sequence identities range between 3 and 20%, i.e. clearly below twilight values for sequence relatedness (25–35%) (26.). The closest structural pairs are aeruginolysin and MMP-8 (Z-score = 15.8) (supplemental Fig. 2 and supplemental Refs.1.and2.),
MMP-8 and snapalysin (15.2), adamalysin II and ulilysin (14.4), and
adamalysin II and leishmanolysin (14.3). The most distant pairs are
astacin and adamalysin II (Z-score = 2.9), MMP-8 and ulilysin (6..2), and snapalysin and ulilysin (6.6).
Distinguishing Features of Each Family
Astacin is a 200-residue digestive enzyme from the crayfish A. astacus and the first member of the astacin family to be structurally analyzed (9.).
Through protein degradation, growth factor activation, extracellular
matrix turnover, and extracellular coat degradation (hatching), astacins
participate in diverse biological processes such as digestion,
development, and tissue remodeling and differentiation (e.g. promoting cartilage and bone formation and collagen biosynthesis) (27.).The three-dimensional structure of astacin shows a packman-like spherical shape and two subdomains of approximately equal size (supplemental Fig. 1A) (9.).
Whereas this proteinase contains only a propeptide and a catalytic MP
domain, most other astacins display additional C-terminal MATH, MAM,
CUB-like, Ser/Thr-rich, I (inserted)-, epidermal growth factor-like,
Tox1, and transmembrane modules (for details, see Refs.
8.,27., and28.).
The astacin family has the longest region of the CSD lacking regular
secondary structure elements, just a small helix and a short β-ribbon,
although this is the sequentially most conserved connecting segment
among metzincins (see Table 1 in Ref.7.). The protein scaffold is cross-linked by four conserved cysteine residues forming two disulfide bridges (supplemental
Fig. 1). The first two residues of the mature enzyme are buried in an
internal cavity in the CSD, and the N-terminal α-amino group establishes
an interaction with a conserved glutamate next to the third
zinc-binding histidine. The unbound coordination of the catalytic zinc
is trigonal-bipyramidal due to the presence of the fourth invariant,
although somewhat more distal, tyrosine zinc ligand downstream of the
Met-turn (supplemental Fig. 1A).
In addition to astacin, the structures of human tolloid-like protease-1
and bone morphogenetic protein-1 have recently been reported (29.).
Serralysins are ∼50-kDa bacterial virulence factors secreted as autoactivatable zymogens by pathogenic γ-class proteobacteria (30.).
These organisms are responsible for human diseases such as meningitis,
endocarditis, pyelonephritis, plague, dermatitis, soft tissue
infections, septicemia, melioidosis, pneumonia, and other respiratory
and urinary tract infections. They play a major role in
hospital-acquired infections due to their capacity to produce surgical
wound infections and to infect neonates. As part of the virulence
potential of these bacteria, serralysins are directed against
coagulation factors and defense-oriented proteins, protease inhibitors,
lysozyme, and transferrin and may cause an anaphylactic response.
The first serralysin to be biochemically and structurally characterized was P. aeruginosa aeruginolysin (
10.).
Its mature 220-residue catalytic domain lacks disulfide connections and
is flanked on its C-terminal end by a calcium-stabilized β-roll domain.
As in astacin, its two subdomains are of similar size. The polypeptide
chain starts with an α-helix in the CSD (characteristic for the family)
that is anchored to the molecular body by a conserved salt bridge with
the C-terminal helix αC (supplemental Fig. 1B).
The NSD features a flap made up by an elongated LβIαA and runs across
the convex surface of the β-sheet. This flap varies greatly among
serralysins (20 reported structures) (supplemental
Table 1), distinctly affecting substrate binding. The CSD of
aeruginolysin presents an extra α-helix within the segment linking the
Met-turn with the wall-forming stretch and a second flap shaped by
residues of the connecting segment. These elements also modulate
substrate binding. Comparison of aeruginolysin, which was first solved
in complex with a bound tetrapeptide (10.), with the closely related structure of unbound Serratia marcescens
serralysin reveals that the unliganded zinc coordination is similar to
astacin (trigonal-bipyramidal). It also includes a tyrosine that
undergoes a hinge motion upon substrate binding (31.).
S. caespitosus
snapalysin is a secreted neutral protease that comprises a 132-residue
catalytic domain preceded by an alanine-rich 100-amino acid N-terminal
extension including a signal peptide and a prodomain. Similar sequences
have been reported for other Streptomyces species, and they have been termed SnpA (Prt and snapalysin), MprA, and SnpA. They show milk-hydrolyzing activity.
Snapalysin
is the smallest metzincin and the only family member that has been
structurally characterized. Its structure recalls a flattened ellipsoid
and bifurcates into two asymmetric subdomains (
12.).
It displays all the characteristic metzincin features, connected by
short loops. Distinguishing elements are a small LβIIβIII protruding
from the upper sheet within the NSD, a small bulge on top of the primed
side of the active-site crevice, a short helix in LβVαB, and a
calcium-binding site (supplemental Fig. 1A).
In addition, an aspartate is found at the position of the third
zinc-binding histidine, and two positions ahead in the sequence, a
conserved tyrosine approaches but not binds the metal.
MMPs
are secreted or membrane-bound proteinases discovered 47 years ago and
participate in tail resorption during tadpole-to-frog metamorphosis.
They are found mainly in higher mammals, although related sequences have
been found in fish, amphibians, insects, plants, prokaryotes, and
viruses. Through turnover of extracellular matrix proteins, MMPs are
involved in tissue resorption, remodeling, and repair, as observed
during embryogenesis and development, branching and organ morphogenesis,
and angiogenesis. However, their potent proteolytic potential or its
absence may also lead to pathologies such as inflammation, ulcers,
rheumatoid arthritis and osteoarthritis, periodontitis, heart failure
and cardiovascular disease, fibrosis, emphysema, and cancer and
metastasis (32.).
More recently, MMPs have been observed to be engaged in (in)activation
events following limited proteolysis, as observed in apoptosis and
intestinal defense protein activation but also in pathologies including
stroke, human immunodeficiency virus-associated dementia,
atherosclerosis, multiple sclerosis, bacterial meningitis, and Alzheimer
disease. MMPs include extracellular proteins such as other
(pro)proteinases, inhibitors, clotting factors, antimicrobial peptides,
and chemotactic and adhesion molecules. In common with ADAMs (see
below), MMPs are also involved in ectodomain shedding of growth factors,
growth factor-binding proteins, hormones and hormone receptors, and
cytokines and cytokine receptors from the cell surface (33.).
Like
other metzincin families, MMPs are mosaic proteins constituted by a
series of inserts and domains. These may include an ∼20-residue
secretory signal peptide, an ∼80-residue propeptide, a 160–170-residue
zinc- and calcium-dependent catalytic proteinase domain, a linker
region, and a 4-fold propeller hemopexin-like C-terminal domain. Further
insertions may include fibronectin type II-related domains; a collagen
type V-like and vitronectin-like insertion domain; a cysteine-rich, a
proline-rich, and an interleukin-1 receptor-like domain; an
immunoglobulin-like domain; a glycosylphosphatidylinositol linkage
signal; a membrane anchor; and a cytoplasmic tail. Naming of MMPs
started historically with fibroblast collagenase as MMP-1 and has
currently reached MMP-28, with 23 different forms described in humans (34.). Those MMPs encompassing a membrane anchor gave rise to the membrane-type MMP subfamily (16.,32.).
Zymogen activation proceeds in MMPs according to a cysteine switch or
Velcro mechanism. This removes the prodomain and switches from an
inactive state, where the Sγ atom of a cysteine residue
within a conserved motif, PRCGVPD, substitutes the catalytic solvent
molecule in the zinc coordination sphere, to the fully accessible active
enzyme (35.,36.). MMPs are the structurally most thoroughly studied metzincin family, with >120 structures reported (supplemental Table 1) (37.). The mature catalytic domain of human neutrophil collagenase (MMP-8) (13.,37.)
has a shallow active-site cavity, which separates a larger NSD (∼120
residues) from a smaller CSD (∼40 amino acids), and generally a deep
hydrophobic S1′ pocket. No disulfide bonds are present in the
structure. The N-terminal α-amino group is anchored to the first of two
conserved aspartates imbedded in helix αC. The NSD displays an S-shaped
double loop connecting strands βIII and βIV, which embraces a
structural zinc cation and a tightly bound calcium ion. The downstream
residues of this segment form a prominent bulge that protrudes into the
active-site groove. LβIVβV and LβIIβIII contribute to a second
calcium-binding site on top of the NSD β-sheet (supplemental Fig. 1, A and B). In the CSD, the MMP-8 chain displays the shortest and most conserved connecting segment within metzincins.
ADAMs/adamalysins/reprolysins
split into three subgroups,
the snake venom MPs,
the mammalian ADAMs,
and
The former are responsible for post-envenomation hemorrhage through
digestion of extracellular matrix components surrounding capillaries,
resulting in tissue necrosis.
In turn, ADAMs were originally described
to play a role in fertilization and sperm function in mammalian
reproductive tracts. They are involved in myogenesis, development,
neurogenesis, differentiation of osteoblastic cells, cell migration
modulation, and muscle fusion. They are also engaged in human disorders
like asthma, cardiac hypertrophy, obesity-associated adipogenesis and
cachexia, rheumatoid arthritis, endotoxic shock, inflammation, and
Alzheimer disease. They also have a major role in protein ectodomain
shedding as described previously for MMPs.
Finally, some family members
lacking the transmembrane domain and harboring multiple copies of a
thrombospondin 1-like repeat and a CUB domain gave rise to a distinct
subfamily of soluble extracellular proteases, the ADAMTSs (42.).
These enzymes disable cell adhesion by binding to integrins. They are
also involved in gonad formation, embryonic development and
angiogenesis, and procollagen activation, as well as in inflammatory
processes, cartilage (aggrecan) degradation in arthritic diseases,
bleeding disorders, and glioma tumor invasion.
All
ADAMs/adamalysins/reprolysins are extracellular multidomain proteins
containing a catalytic zinc- and calcium-dependent MP domain. In
addition, they can display a prodomain and C-terminal disintegrin-like,
cysteine-rich, C-type lectin, epidermal growth factor-like,
thrombospondin 1-like, and/or transmembrane domains, as well as a
cytoplasmic domain. Latency is maintained by the prodomain, and
activation is believed to occur as in MMPs, i.e. by cleavage of the prodomain according to a cysteine switch-like mechanism (7.,36.,43.).
The first catalytic domain structure to be analyzed was that of adamalysin II from C. adamanteus snake venom (11.).
This is a compact 203-residue molecule of oblate ellipsoidal shape,
notched at the periphery to render a relatively flat substrate-fixing
cleft. This cleft separates a large ∼150-residue NSD from a small
∼50-residue CSD (supplemental Fig. 1A). Both the N and C termini are surface-located; the former is linked by a salt bridge to the C-terminal helix αC (7.).
Adamalysin II deviates most from the metzincin consensus sequence
within the conserved regular secondary structure elements, especially at
strand βI and helices αA and αC (Fig. 1B).
Inserted into the common scaffold, two additional helices are found
within the NSD. In the CSD, two disulfide bonds cross-link the irregular
connecting segment and attach helix αC to the NSD, respectively. A
calcium ion is located on the surface, opposite the active site and
close to the C terminus (supplemental Fig. 1A). The S1′
pocket, characterized by a pronounced bulge segment LβIVβV, is
hydrophobic and deep, reminiscent of some MMPs.
In addition to
adamalysin II, a number of snake venom MPs (ADAM-17, ADAM-33, and
ADAMTS-1, -4, and -5) have been structurally analyzed to date (supplemental Table 1).
Leishmanolysins
are cell-surface proteins present in most trypanosomatid, plasmodiid,
and sarcocystid protozoa. They constitute the major component of the
promastigote surface and are enzymatically active against polypeptide
substrates. They cleave CD4 molecules at the surface of human T cells
and protect promastigotes from lysis by complement proteins, suggesting a
possible role as a virulence factor. Related sequences have been found
in mammals (here called invadolysin), fruit flies, thale cress,
nematodes, and bacteria (7.,44.).
The only structurally analyzed family member is L. major
leishmanolysin. It is synthesized as a 602-residue inactive precursor
in the endoplasmic reticulum with a signal and a 100-residue propeptide,
which includes a highly conserved cysteine residue potentially acting
as a cysteine switch (see above). Activation liberates a mature MP of
∼280 residues, followed by an ∼200-residue C-terminal domain. A
63-residue insertion domain is observed between the glycine and the
third zinc-binding histidine of the long consensus motif (supplemental Fig. 1A).
The MP domain is the most asymmetric among metzincins, with a
175-residue NSD and just an ∼45-amino acid CSD. Its N terminus is
located on the back left surface. The NSD is characterized by a β-sheet,
which lacks strand βII (supplemental Fig. 1, A and B),
and by the presence of two unique ∼40-amino acid inserted flaps, which
account for most of the differences in size from the other proteins of
the clan. The NSD is cross-linked by two disulfide bonds. Preceding
strand βIV, a slightly prominent bulge segment lies on top of the
shallow, medium-sized S1′ pocket, which is delimited by the
wall-forming segment and the beginning of the active-site helix αB. At
the end of the CSD, helix αC is followed by a segment in an extended
conformation, which runs from left to right across the back surface (14.).
The
most recent family to be structurally characterized are the
pappalysins. They were named after human PAPP-A, a heavily glycosylated
170-kDa multidomain protein specifically cleaving insulin-like growth
factor-binding proteins (45.). Proulilysin is a 38-kDa archaeal protein from M. acetivorans that shares sequence similarity with PAPP-A, but it encompasses only the prodomain and the catalytic domain (15.).
The proprotein may undergo cysteine switch-mediated activation, as
suggested by the presence of a conserved cysteine in the prodomain.
Activation occurs autolytically in the presence of calcium.
With 262
residues, mature ulilysin is the largest MP of all metzincin catalytic
domains. As distinguishing features, it presents in the NSD a loop
dividing strand βII into two substrands (βII and βII′) and a β-ribbon
inserted within LβIIIβIV that protrudes from the molecular surface and
frames the active site on its primed side. The segment connecting helix
αA with strand βII is the largest among metzincins and covers almost all
of the back of the molecule from the NSD to the CSD in a cape-like
fashion and includes two unique α-helices. The glycine of the
zinc-binding motif is replaced in ulilysin and a small subset of
pappalysins by an asparagine under slight variation of the main-chain
angles, which do not correspond here to a high-energy conformation but
to a left-handed α-helix. Overall, the chain trace flanking this residue
is indistinguishable from other metzincins (supplemental Fig. 1) (7.).
The CSD shows two disulfide bonds and a unique two-calcium site. This
site is a molecular switch for activity, as the proteinase can be
reversibly inhibited through calcium chelators ( 15.,20.,21.).
Finally, in the absence of an unbound structure, ulilysin may possess a
fifth zinc-binding tyrosine ligand provided by the Met-turn that is
swung out upon substrate binding.
Conclusions
The
metzincins constitute a clan of ubiquitous MPs present in all kingdoms
of life, which are pivotal for physiology and pathology. So far, seven
families have been structurally analyzed. The clan represents a case of
divergent evolution from an urmetzincin, which may not look very
different from the smallest member, snapalysin. Into such a minimal
scaffold, evolution has introduced unique structural elements conserved
among each family. Based on the presence of an extended zinc-binding
consensus sequence pattern, several more families have been suggested to
enlarge the clan, although future structural analysis will be required
to confirm their ascription. Structural information on each of the seven
families may help to elucidate common catalytic and processing
mechanisms and to assign the function of proteins encoded by newly
discovered gene sequences.
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