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1.
Nat Immunol ; 22(6): 757-768, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34031614

RESUMO

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell-dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR-CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.


Assuntos
Linfócitos B/imunologia , Ativação do Complemento , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Centro Germinativo/imunologia , Animais , Animais Geneticamente Modificados , Linfócitos B/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Linhagem Celular Tumoral , Hematopoiese Clonal/imunologia , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Tonsila Palatina/citologia , Tonsila Palatina/patologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/metabolismo
2.
Immunity ; 55(3): 459-474.e7, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35148827

RESUMO

Type I interferons (IFNs) are pleiotropic cytokines with potent antiviral properties that also promote protective T cell and humoral immunity. Paradoxically, type I IFNs, including the widely expressed IFNß, also have immunosuppressive properties, including promoting persistent viral infections and treating T-cell-driven, remitting-relapsing multiple sclerosis. Although associative evidence suggests that IFNß mediates these immunosuppressive effects by impacting regulatory T (Treg) cells, mechanistic links remain elusive. Here, we found that IFNß enhanced graft survival in a Treg-cell-dependent murine transplant model. Genetic conditional deletion models revealed that the extended allograft survival was Treg cell-mediated and required IFNß signaling on T cells. Using an in silico computational model and analysis of human immune cells, we found that IFNß directly promoted Treg cell induction via STAT1- and P300-dependent Foxp3 acetylation. These findings identify a mechanistic connection between the immunosuppressive effects of IFNß and Treg cells, with therapeutic implications for transplantation, autoimmunity, and malignancy.


Assuntos
Interferon beta , Linfócitos T Reguladores , Acetilação , Aloenxertos , Animais , Fatores de Transcrição Forkhead/metabolismo , Sobrevivência de Enxerto , Humanos , Interferon beta/metabolismo , Camundongos
3.
Nature ; 622(7982): 393-401, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37821590

RESUMO

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.


Assuntos
Rejeição de Enxerto , Transplante de Rim , Macaca fascicularis , Suínos , Transplante Heterólogo , Animais , Humanos , Animais Geneticamente Modificados , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Transplante de Rim/métodos , Polissacarídeos/deficiência , Suínos/genética , Transplante Heterólogo/métodos , Transgenes/genética
4.
J Biol Chem ; 298(6): 101985, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35483450

RESUMO

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.


Assuntos
Proteínas de Escherichia coli/química , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Proteínas Periplásmicas/química , Sítios de Ligação , Lectina de Ligação a Manose da Via do Complemento , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lectinas/genética , Lectinas/metabolismo , Lectina de Ligação a Manose/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Periplásmicas/metabolismo , Subunidades Proteicas
5.
PLoS Pathog ; 15(12): e1008232, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31860690

RESUMO

Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin- and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathway-related and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.


Assuntos
Lectina de Ligação a Manose da Via do Complemento/fisiologia , Proteínas do Sistema Complemento/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Periplásmicas/metabolismo , Serina Proteases/sangue , Ativação do Complemento/fisiologia , Escherichia coli/metabolismo , Humanos , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/metabolismo , Yersinia pestis/metabolismo
7.
J Biol Chem ; 292(7): 2690-2702, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28062577

RESUMO

Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.


Assuntos
Evolução Molecular , Isoenzimas/metabolismo , Elastase Pancreática/metabolismo , Sequência de Aminoácidos , Animais , Bacteriófagos/genética , Humanos , Proteínas de Insetos/metabolismo , Isoenzimas/genética , Cinética , Elastase Pancreática/genética , Peptídeos/metabolismo , Especificidade por Substrato , Suínos
8.
Proc Natl Acad Sci U S A ; 109(26): 10498-503, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22691502

RESUMO

The lectin pathway of complement activation is an important component of the innate immune defense. The initiation complexes of the lectin pathway consist of a recognition molecule and associated serine proteases. Until now the autoactivating mannose-binding lectin-associated serine protease (MASP)-2 has been considered the autonomous initiator of the proteolytic cascade. The role of the much more abundant MASP-1 protease was controversial. Using unique, monospecific inhibitors against MASP-1 and MASP-2, we corrected the mechanism of lectin-pathway activation. In normal human serum, MASP-2 activation strictly depends on MASP-1. MASP-1 activates MASP-2 and, moreover, inhibition of MASP-1 prevents autoactivation of MASP-2. Furthermore we demonstrated that MASP-1 produces 60% of C2a responsible for C3 convertase formation.


Assuntos
Ativação do Complemento , Lectinas/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Coagulação Sanguínea , Convertases de Complemento C3-C5/metabolismo , Ativação Enzimática , Humanos , Serina Proteases Associadas a Proteína de Ligação a Manose/antagonistas & inibidores
9.
J Biol Chem ; 288(13): 8922-34, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23386610

RESUMO

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.


Assuntos
Lectina de Ligação a Manose da Via do Complemento , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Catálise , Proteínas do Sistema Complemento , Humanos , Imunidade Inata , Cinética , Lectinas/química , Lectinas de Ligação a Manose/química , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Mutação , Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
10.
J Biol Chem ; 287(24): 20290-300, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22511776

RESUMO

The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.


Assuntos
Lectina de Ligação a Manose da Via do Complemento , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Inibidores de Proteases/química , Cristalografia por Raios X , Humanos , Serina Proteases Associadas a Proteína de Ligação a Manose/antagonistas & inibidores , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
11.
J Biol Chem ; 286(25): 22535-45, 2011 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-21515688

RESUMO

Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2' (Leu or Asp), and P4' (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2' (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4' (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K(D) 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K(D) of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys(51), Arg(56), and Arg(80)) on the surface of human CTRC that interact with the P4' acidic residue of the inhibitor. The acidic preference of CTRC at P4' is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.


Assuntos
Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Biblioteca de Peptídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Quimotripsina/química , Quimotripsina/genética , Análise Mutacional de DNA , Evolução Molecular Direcionada , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Inibidores de Proteases/química , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
12.
Transplant Cell Ther ; 28(8): 472.e1-472.e11, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35643350

RESUMO

Conditioning regimens used for hematopoietic stem cell transplantation (HCT) can escalate the severity of acute T cell-mediated graft-versus-host disease (GVHD) by disrupting gastrointestinal integrity and initiating lipopolysaccharide (LPS)-dependent innate immune cell activation. Activation of the complement cascade has been associated with murine GVHD, and previous work has shown that alternative pathway complement activation can amplify T cell immunity. Whether and how mannan-binding lectin (MBL), a component of the complement system that binds mannose as well as oligosaccharide components of LPS and lipoteichoic acid, affects GVHD is unknown. In this study, we tested the hypothesis that MBL modulates murine GVHD and examined the mechanisms by which it does so. We adoptively transferred C3.SW bone marrow (BM) cells ± T cells into irradiated wild type (WT) or MBL-deficient C57Bl/6 (B6) recipients with or without inhibiting MBL-initiated complement activation using C1-esterase inhibitor (C1-INH). We analyzed the clinical severity of disease expression and analyzed intestinal gene and cell infiltration. In vitro studies assessed MBL expression on antigen-presenting cells (APCs) and compared LPS-induced responses of WT and MBL-deficient APCs. MBL-deficient recipients of donor BM ± T cells exhibited significantly less weight loss over the first 2 weeks post-transplantation weeks compared with B6 controls (P < .05), with similar donor engraftment in the 2 groups. In recipients of C3.SW BM + T cells, the clinical expression of GVHD was less severe (P < .05) and overall survival was better (P < .05) in MBL-deficient mice compared with WT mice. On day-7 post-transplantation, analyses showed that the MBL-deficient recipients exhibited less intestinal IL1b, IL17, and IL12 p40 gene expression (P < .05 for each) and fewer infiltrating intestinal CD11c+, CD11b+, and F4/80+ cells and TCRß+, CD4+, CD4+IL17+, and CD8+ T cells (P < .05 for each). Ovalbumin or allogeneic cell immunizations induced equivalent T cell responses in MBL-deficient and WT mice, demonstrating that MBL-deficiency does not directly impact T cell immunity in the absence of irradiation conditioning. Administration of C1-INH did not alter the clinical expression of GVHD in preconditioned WT B6 recipients, suggesting that MBL amplifies clinical expression of GVHD via a complement-independent mechanism. WT, but not MBL-deficient, APCs express MBL on their surfaces. LPS-stimulated APCs from MBL-deficient mice produced less proinflammatory cytokines (P < .05) and induced weaker alloreactive T cell responses (P < .05) compared with WT APCs. Together, our data show that MBL modulates murine GVHD, likely by amplifying complement-independent, LPS-initiated gastrointestinal inflammation. The results suggest that devising strategies to block LPS/MBL ligation on APCs has the potential to reduce the clinical expression of GVHD.


Assuntos
Doença Enxerto-Hospedeiro , Inflamação , Lectina de Ligação a Manose , Animais , Transplante de Medula Óssea , Linfócitos T CD8-Positivos , Doença Enxerto-Hospedeiro/genética , Inflamação/etiologia , Inflamação/genética , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Lectina de Ligação a Manose/genética , Camundongos , Camundongos Endogâmicos C57BL , Transplante Homólogo
13.
J Mol Biol ; 431(3): 557-575, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30543823

RESUMO

Reversible serine proteinase inhibitors comprise 18 unrelated families. Each family has a distinct representative structure but contains a surface loop that adopts the same, canonical conformation in the enzyme-inhibitor complex. The Laskowski mechanism universally applies for the action of all canonical inhibitors independent of their scaffold, but it has two nontrivial extrapolations. Intrascaffolding additivity states that all enzyme-contacting loop residues act independently of each other, while interscaffolding additivity claims that these residues act independently of the scaffold. These theories have great importance for engineering proteinase inhibitors but have not been comprehensively challenged. Therefore, we tested the interscaffolding additivity theory by hard-randomizing all enzyme-contacting canonical loop positions of a Kazal- and a Pacifastin-scaffold inhibitor, displaying the variants on M13 phage, and selecting the libraries on trypsin and chymotrypsin. Directed evolution delivered different patterns on both scaffolds against both enzymes, which contradicts interscaffolding additivity. To quantitatively assess the extent of non-additivity, we measured the affinities of the optimal binding loop variants and their binding loop-swapped versions. While optimal variants have picomolar affinities, swapping the evolved loops results in up to 200,000-fold affinity loss. To decipher the underlying causes, we characterized the stability, overall structure and dynamics of the inhibitors with differential scanning calorimetry, circular dichroism and NMR spectroscopy and molecular dynamic simulations. These studies revealed that the foreign loop destabilizes the lower-stability Pacifastin scaffold, while the higher-stability Kazal scaffold distorts the foreign loop. Our findings disprove interscaffolding additivity and show that loop and scaffold form one integrated unit that needs to be coevolved to provide high-affinity inhibition.


Assuntos
Inibidores de Serina Proteinase/química , Sítios de Ligação , Varredura Diferencial de Calorimetria/métodos , Quimotripsina/química , Dicroísmo Circular/métodos , Humanos , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Serina Proteases/química , Tripsina/química
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