Документ взят из кэша поисковой машины. Адрес оригинального документа : http://kodomo.cmm.msu.ru/~lesya/Zachot.doc Дата изменения: Mon Mar 23 10:17:29 2009 Дата индексирования: Mon Oct 1 22:29:26 2012 Кодировка: koi8-r

Информация о структуре и особенностях белка Екотина.

Екотин представляет собой гомодимерный белок,составленный из 162
аминокислотных остатков,локализованный в периплазме клетки E.coli.
Обнаружен он у протобактерий, гаммапротобактерий. Его функцией является
ингибирование деятельности панкреотических протеаз,таких как
трипсин,химотрипсин,эластаза,
калликреин. Серин-протеазы очень интересны,потому что обеспечивают
детализированное понимание своих ферментативных свойств и своей роли во
многих физиологических и патологических процессах. При проведении ряда
исследований удалось развить макромолекулярный ингибитор Екотин,который
является таким образом специализированным белком-ингибитором для серин
протеаз. Ингибирование деятельности протеазы с помощью дикого типа и
спроектированного Екотина приводит к замедленному развитию простаты крысы и
человеческих простатических опухолей.
Екотин уникален в своей способности и механизме ингибирования серин-протеаз
широкого специфического диапазона. Екотин связывается с человеческим альфа-
тромбином через его вторичый связывающий участок,провоцирует "удаление"
двух активаторов,в результате происходит существенная задержка исчеления
раны кожи.
Он является маленьким периплазмаическим белком-16 kDa,содержит одну
двусернистую облигацию. Дисульфидные мостики образуются между боковыми
цепями 70-го и 107-го аминокислотного остатка.Их образует цистеин. Екотин
принадлежит семейству Екотин-ингибиторам протеаз I11.Первые 20 остатков
выполняют сигнальную функцию.

Идентификаторы: Ecotin, eco, eti,b2209,JW2197,P23827.

Последовательность аминокислот в фаста-формате:

>Eco_ECOLI ecotin precursor
MKTILPAVLFAAFATTSAWAAESVQPLEKI
APYPQAEKGMKRQVIQLTPQEDESTLKVEL
LIGQTLEVDCNLHRLGGKLENKTLEGWGYD
YYVFDKVSSPVSTMMACPDGKKEKKFVTAY
LGDAGMLRYNSKLPIVVYTPDNVDVKYRVW
KAEEKIDNAVVR

Из белков,схожих с Екотином функциями,только ещё один имет третичную
структуру.
Третичная структура екотина выглядит следующим обазом:
Структура включает в себя:1 альфа-спираль;10 бета-тяжей;1 бета-поворот.
[pic]


1."The sequence and reactive site of ecotin. A general inhibitor of
pancreatic serine proteases from Escherichia coli."
http://www.jbc.org/cgi/reprint/266/10/6620
2."Molecular cloning of the ecotin gene in Escherichia coli."
The nucleotide sequence of a 876 bp region in E. coli chromosome that
encodes Ecotin was determined. The proposed coding sequence for Ecotin is
486 nucleotides long, which would encode a protein consisting of 162 amino
acids with a calculated molecular weight of 18,192 Da. The deduced primary
sequence of Ecotin includes a 20-residue signal sequence, cleavage of which
would give rise to a mature protein with a molecular weight of 16,099 Da.
Ecotin does not contain any consensus reactive site sequences of known
serine protease inhibitor families, suggesting that Ecotin is a novel
inhibitor.

3."Automated multiplex sequencing of the E.coli genome."

4."A 460-kb DNA sequence of the Escherichia coli K-12 genome corresponding
to the 40.1-50.0 min region on the linkage map."

http://dnaresearch.oxfordjournals.org/cgi/reprint/3/6/379
5."The complete genome sequence of Escherichia coli K-12."

The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of
4288 protein-coding genes annotated, 38 percent have no attributed
function. Comparison with five other sequenced microbes reveals ubiquitous
as well as narrowly distributed gene families; many families of similar
genes within E. coli are also evident. The largest family of paralogous
proteins contains 80 ABC transporters. The genome as a whole is strikingly
organized with respect to the local direction of replication; guanines,
oligonucleotides possibly related to replication and recombination, and
most genes are so oriented. The genome also contains insertion sequence
(IS) elements, phage remnants, and many other patches of unusual
composition indicating genome plasticity through horizontal transfer.

6."Highly accurate genome sequences of Escherichia coli K-12 strains MG1655
and W3110."

http://www.nature.com/msb/journal/v2/n1/full/msb4100049.html
7. "Comparing the predicted and observed properties of proteins encoded in
the genome of Escherichia coli K-12."

Mining the emerging abundance of microbial genome sequences for hypotheses
is an exciting prospect of "functional genomics". At the forefront of this
effort, we compared the predictions of the complete Escherichia coli
genomic sequence with the observed gene products by assessing 381 proteins
for their mature N-termini, in vivo abundances, isoelectric points,
molecular masses, and cellular locations. Two-dimensional gel
electrophoresis (2-DE) and Edman sequencing were combined to sequence
Coomassie-stained 2-DE spots representing the abundant proteins of wild-
type E. coli K-12 strains. Greater than 90% of the abundant proteins in the
E. coli proteome lie in a small isoelectric point and molecular mass window
of 4-7 and 10-100 kDa, respectively. We identified several highly abundant
proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been
predicted from the genomic sequence alone. Of the 223 uniquely identified
loci, 60% of the encoded proteins are proteolytically processed. As
previously reported, the initiator methionine was efficiently cleaved when
the penultimate amino acid was serine or alanine. In contrast, when the
penultimate amino acid was threonine, glycine, or proline, cleavage was
variable, and valine did not signal cleavage. Although signal peptide
cleavage sites tended to follow predicted rules, the length of the putative
signal sequence was occassionally greater than the consensus. For proteins
predicted to be in the cytoplasm or inner membrane, the N-terminal amino
acids were highly constrained compared to proteins localized to the
periplasm or outer membrane. Although cytoplasmic proteins follow the N-end
rule for protein stability, proteins in the periplasm or outer membrane do
not follow this rule; several have N-terminal amino acids predicted to
destabilize the proteins. Surprisingly, 18% of the identified 2-DE spots
represent isoforms in which protein products of the same gene have
different observed pI and M(r), suggesting they are post-translationally
processed. Although most of the predicted and observed values for
isoelectric point and molecular mass show reasonable concordance, for
several proteins the observed values significantly deviate from the
expected values. Such discrepancies may represent either highly processed
proteins or misinterpretations of the genomic sequence. Our data suggest
that AhpC, CspC, and HdeA exist as covalent homomultimers, and that IcdA
exists as at least three isoforms even under conditions in which covalent
modification is not predicted. We enriched for proteins based on
subcellular location and found several proteins in unexpected subcellular
locations.
8. "Macromolecular chelation as an improved mechanism of protease
inhibition: structure of the ecotin-trypsin complex."
The 2.4 A crystal structure (R = 0.180) of the serine protease inhibitor
ecotin was determined in a complex with trypsin. Ecotin's dimer structure
provides a second discrete and distal binding site for trypsin and, as
shown by modelling experiments, other serine proteases. The second site is
approximately 45 A from the reactive/active site of the complex and
features 13 hydrogen bonds, including six that involve carbonyl oxygen
atoms and four bridged by water molecules. Contacts ecotin makes with
trypsin's active site are similar to, though more extensive than, those
found between trypsin and basic pancreatic trypsin inhibitor. The side
chain of ecotin Met84 is found in the substrate binding pocket of trypsin
where it makes few contacts, but also does not disrupt the solvent
structure or cause misalignment of the scissile bond. This first case of
protein dimerization being used to augment binding energy and allow
chelation of a target protein provides a new model for protein-protein
interactions and for protease inhibition.
9. "Crystal structure analyses of uncomplexed ecotin in two crystal forms:
implications for its function and stability."
Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel
serine protease inhibitor present in Escherichia coli. Its thermostability
and acid stability, as well as broad specificity toward proteases, make it
an interesting protein for structural characterization. Its structure in
the uncomplexed state, determined for two different crystalline
environments, allows a structural comparison of the free inhibitor with
that in complex with trypsin. Although there is no gross structural
rearrangement of ecotin when binding trypsin, the loops involved in binding
trypsin show relatively large shifts in atomic positions. The inherent
flexibility of the loops and the highly nonglobular shape are the two
features essential for its inhibitory function. An insight into the
understanding of the structural basis of thermostability and acid stability
of ecotin is also provided by the present structure.
10. "Crystal structure of an ecotin-collagenase complex suggests a model
for recognition and cleavage of the collagen triple helix."
The crystal structure of fiddler crab collagenase complexed with the
dimeric serine protease inhibitor ecotin at 2.5 A resolution reveals an
extended cleft providing binding sites for at least 11 contiguous substrate
residues. Comparison of the positions of nine intermolecular main chain
hydrogen bonding interactions in the cleft, with the known sequences at the
cleavage site of type I collagen, suggests that the protease binding loop
of ecotin adopts a conformation mimicking that of the cleaved strand of
collagen. A well-defined groove extending across the binding surface of the
enzyme readily accommodates the two other polypeptide chains of the triple-
helical substrate. These observations permit construction of a detailed
molecular model for collagen recognition and cleavage by this invertebrate
serine protease. Ecotin undergoes a pronounced internal structural
rearrangement which permits binding in the observed conformation. The
capacity for such rearrangement appears to be a key determinant of its
ability to inhibit a wide range of serine proteases
11. "Compromise and accommodation in ecotin, a dimeric macromolecular
inhibitor of serine proteases."
Ecotin is a dimeric serine protease inhibitor from Escherichia coli which
binds proteases to form a hetero-tetramer with three distinct interfaces:
an ecotin-ecotin dimer interface, a larger primary ecotin-protease
interface, and a smaller secondary ecotin-protease interface. The
contributions of these interfaces to binding and inhibition are unequal. To
investigate the contribution and adaptability of each interface, we have
solved the structure of two mutant ecotin-trypsin complexes and compared
them to the structure of the previously determined wild-type ecotin-trypsin
complex. Wild-type ecotin has an affinity of 1 nM for trypsin, while the
optimized mutant, ecotin Y69F, D70P, which was found using phage display
technologies, inhibits rat trypsin with a K(i) value of 0.08 nM. Ecotin 67-
70A, M84R which has four alanine substitutions in the ecotin-trypsin
secondary binding site, along with the M84R mutation at the primary site,
has a K(i) value against rat trypsin of 0.2 nM. The structure of the ecotin
Y69F, D70P-trypsin complex shows minor structural changes in the ecotin-
trypsin tetramer. The structure of the ecotin 67-70A, M84R mutant bound to
trypsin shows large deviations in the tertiary and quaternary structure of
the complex. The trypsin structure shows no significant changes, but the
conformation of several loop regions of ecotin are altered, resulting in
the secondary site releasing its hold on trypsin. The structure of several
regions previously considered to be rigid is also significantly modified.
The inherent flexibility of ecotin allows it to accommodate these mutations
and still maintain tight binding through the compromises of the protein-
protein interfaces in the ecotin-trypsin tetramer. A comparison with two
recently described ecotin-like genes from other bacteria suggests that
these structural and functional features are conserved in otherwise distant
bacterial lineages. Copyright 2000 Academic Press.
12. "Crystal structure of thrombin-ecotin reveals conformational changes
and extended interactions."
The protease inhibitor ecotin fails to inhibit thrombin despite its broad
specificity against serine proteases. A point mutation (M84R) in ecotin
results in a 1.5 nM affinity for thrombin, 10(4) times stronger than that
of wild-type ecotin. The crystal structure of bovine thrombin is determined
in complex with ecotin M84R mutant at 2.5 A resolution. Surface loops
surrounding the active site cleft of thrombin have undergone significant
structural changes to permit inhibitor binding. Particularly, the insertion
loops at residues 60 and 148 in thrombin, which likely mediate the
interactions with macromolecules, are displaced when the complex forms.
Thrombin and ecotin M84R interact in two distinct surfaces. The loop at
residue 99 and the C-terminus of thrombin contact ecotin through mixed
polar and nonpolar interactions. The active site of thrombin is filled with
eight consecutive amino acids of ecotin and demonstrates thrombin's
preference for specific features that are compatible with the thrombin
cleavage site: negatively charged-Pro-Val-X-Pro-Arg-hydrophobic-positively
charged (P1 Arg is in bold letters). The preference for a Val at P4 is
clearly defined. The insertion at residue 60 may further affect substrate
binding by moving its adjacent loops that are part of the substrate
recognition sites.
13. "Ecotin: lessons on survival in a protease-filled world."
Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has
been shown to be a potent inhibitor of a group of homologous serine
proteases with widely differing substrate recognition. It is highly
effective against a number of enzymes, including both pancreatic and
neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and
kallikrein. Recent structural and functional studies on ecotin and its
interactions with different serine proteases have clarified these initial
observations and revealed the remarkable features of this protein in
inhibiting a strikingly large subset of the chymotrypsin family of serine
proteases. The structures of the ecotin:serine protease complexes provide
the first examples of protein-protein recognition where the concept of
specificity of interactions needs to be reexamined. The binding sites show
a fluidity of protein contacts derived from ecotin's innate flexibility in
fitting itself to proteases while strongly interfering with their function.
Всего о белке найдено 13 публикаций.