Modulation of Integrin Activation by an Entropic Spring in the β-Knee

We show that the length of a loop in the β-knee, between the first and second cysteines (C1-C2) in integrin EGF-like (I-EGF) domain 2, modulates integrin activation. Three independent sets of mutants, including swaps among different integrin β-subunits, show that C1-C2 loop lengths of 12 and longer favor the low affinity state and masking of ligand-induced binding site (LIBS) epitopes. Shortening length from 12 to 4 residues progressively increases ligand binding and LIBS epitope exposure. Compared with length, the loop sequence had a smaller effect, which was ascribable to stabilizing loop conformation, and not interactions with the α-subunit. The data together with structural calculations support the concept that the C1-C2 loop is an entropic spring and an emerging theme that disordered regions can regulate allostery. Diversity in the length of this loop may have evolved among integrin β-subunits to adjust the equilibrium between the bent and extended conformations at different set points.     

Intracellular signals direct integrin localization to sites of function in embryonic muscles

In the Drosophila embryo, the alphaPS2betaPS integrin heterodimer is localized tightly at the termini of the multinucleate muscles where they attach to the alphaPS1betaPS-containing epidermal tendon cells. Here we examine the basis for alphaPS2betaPS integrin subcellular localization. We show that the betaPS cytoplasmic tail is sufficient to direct the localization of a heterologous transmembrane protein, CD2, to the muscle termini in vivo. This localization does not occur via an association with structures set up by the endogenous betaPS integrins, since it can occur even in the absence of the betaPS protein. Furthermore, the subcellular localization of the alphaPS2betaPS integrin is not dependent on any other interactions between the muscles and the tendon cells. In embryos that lack the segmental tendon cells, due to a mutation removing the related segment polarity genes engrailed and invected, alphaPS2betaPS is still localized to the muscle termini even though the ventral longitudinal muscles are not attached to the epidermis, but instead are attached end to end. Thus the alphaPS2betaPS integrin can be localized by an intracellular mechanism within the muscles. Our results challenge the view that the transmission of signals from the extracellular environment via integrins is required for the organization of the cytoskeleton and the resultant cellular polarity.     

Mutational analysis of MAdCAM-1/alpha4beta7 interactions reveals significant binding determinants in both the first and second immunuglobulin domains

The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin. MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin alpha4beta7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin alpha4beta7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/alpha4beta7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.     

Amino acid changes in Drosophila alphaPS2betaPS integrins that affect ligand affinity

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2betaPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavbeta3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2betaPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the betaPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the betaPS I domain (betaPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2betaPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin beta subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the betaPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because betaPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.     

NMR studies of a DNA containing 8-hydroxydeoxyguanosine.

The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.     

Mutational analysis of MAdCAM-1/α4β7 interactions reveals significant binding determinants in both the first and second immunoglobulin domains

The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin, MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin α4β7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin α4β7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/α4β7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.     

Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly

The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.     

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (DLG), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (FAS2) and the position specific (PS) integrin receptors, including betaPS and its alpha partners (alphaPS1, alphaPS2, alphaPS3/alphaVolado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that DLG and FAS2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. We show that PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. betaPS integrin physically associates with the synaptic complex anchored by the DLG scaffolding protein, which contains CaMKII and FAS2. We demonstrate an alteration of the FAS2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory betaPS integrin mutations increase the expression and synaptic localization of FAS2. Synaptic structural defects in betaPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of FAS2 (distal in pathway). These studies demonstrate that betaPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology.     

Identification of integrin beta subunit mutations that alter heterodimer function in situ

We conducted a genetic screen for mutations in myospheroid, the gene encoding the Drosophila betaPS integrin subunit, and identified point mutants in all of the structural domains of the protein. Surprisingly, we find that mutations in very strongly conserved residues will often allow sufficient integrin function to support the development of adult animals, including mutations in the ADMIDAS site and in a cytoplasmic NPXY motif. Many mutations in the I-like domain reduce integrin expression specifically when betaPS is combined with activating alphaPS2 cytoplasmic mutations, indicating that integrins in the extended conformation are unstable relative to the inactive, bent heterodimers. Interestingly, the screen has identified alleles that show gain-of-function characteristics in cell culture, but have negative effects on animal development or viability. This is illustrated by the allele mys(b58); available structural models suggest that the molecular lesion of mys(b58), V409>D, should promote the "open" conformation of the beta subunit I-like domain. This expectation is supported by the finding that alphaPS2betaPS (V409>D) promotes adhesion and spreading of S2 cells more effectively than does wild-type alphaPS2betaPS, even when betaPS is paired with alphaPS2 containing activating cytoplasmic mutations. Finally, comparisons with the sequence of human beta8 suggest that evolution has targeted the "mys(b58)" residue as a means of affecting integrin activity.     

Identification of integrin beta subunit mutations that alter affinity for extracellular matrix ligand

We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.     

Modulation of ligand binding by alternative splicing of the alphaPS2 integrin subunit

The Drosophila alphaPS2 integrin subunit is found in two isoforms. alphaPS2C contains 25 residues not found in alphaPS2m8, encoded by the alternative eighth exon. Previously, it was shown that cells expressing alphaPS2C spread more effectively than alphaPS2m8 cells on fragments of the ECM protein Tiggrin, and that alphaPS2C-containing integrins are relatively insensitive to depletion of Ca(2+). Using a ligand mimetic probe for Tiggrin affinity (TWOW-1), we show that the affinity of alphaPS2CbetaPS for this ligand is much higher than that of alphaPS2m8betaPS. However, the two isoforms become more similar in the presence of activating levels of Mn(2+). Modeling indicates that the exon 8-encoded residues replace the third beta strand of the third blade of the alpha subunit beta-propeller structure, and generate an exaggerated loop between this and the fourth strand. alphaPS2 subunits with the extra loop structure but with an m8-like third strand, or subunits with a C-like strand but an m8-like short loop, both fail to show alphaPS2C-like affinity for TWOW-1. Surprisingly, a single C > m8-like change at the third strand-loop transition point is sufficient to make alphaPS2C require Ca(2+) for function, despite the absence of any known cation binding site in this region. These data indicate that alternative splicing in integrin alpha subunit extracellular domains may affect ligand affinity via relatively subtle alterations in integrin conformation. These results may have relevance for vertebrate alpha6 and alpha7, which are alternatively spliced at the same site.     

Integrins modulate Sog activity in the Drosophila wing

Morphogenesis of the Drosophila wing depends on a series of cell-cell and cell-extracellular matrix interactions. During pupal wing development, two secreted proteins, encoded by the short gastrulation (sog) and decapentaplegic (dpp) genes, vie to position wing veins in the center of broad provein territories. Expression of the Bmp4 homolog dpp in vein cells is counteracted by expression of the Bmp antagonist sog in intervein cells, which results in the formation of straight veins of precise width. We screened for genetic interactions between sog and genes encoding a variety of extracellular components and uncovered interactions between sog and myospheroid (mys), multiple edematous wing (mew) and scab (scb), which encode betaPS, alphaPS1 and alphaPS3 integrin subunits, respectively. Clonal analysis reveals that integrin mutations affect the trajectory of veins inside the provein domain and/or their width and that misexpression of sog can alter the behavior of cells in such clones. In addition, we show that a low molecular weight form of Sog protein binds to alphaPS1betaPS. We find that Sog can diffuse from its intervein site of production into adjacent provein domains, but only on the dorsal surface of the wing, where Sog interacts functionally with integrins. Finally, we show that Sog diffusion into provein regions and the reticular pattern of extracellular Sog distribution in wild-type wings requires mys and mew function. We propose that integrins act by binding and possibly regulating the activity/availability of different forms of Sog during pupal development through an adhesion independent mechanism.     

Genetic analysis of the Drosophila alphaPS2 integrin subunit reveals discrete adhesive,morphogenetic and sarcomeric functions.

The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role of integrins, but evidence for integrin signalling has remained elusive. Each integrin is an alphabeta heterodimer, and the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integrin expression includes, but is more extensive than, the sites where PS2 has a known requirement. In order to investigate whether PS2 integrin is required at these additional sites and/or has functions besides mediating adhesion, a comprehensive genetic analysis of inflated, the gene that encodes alphaPS2, was performed. We isolated 35 new inflated alleles, and obtained 10 alleles from our colleagues. The majority of alleles are amorphs (36/45) or hypomorphs (4/45), but five alleles that affect specific developmental processes were identified. Interallelic complementation between these alleles suggests that some may affect distinct functional domains of the alphaPS2 protein, which specify particular interactions that promote adhesion or signalling. One new allele reveals that the PS2 integrin is required for the development of the adult halteres and legs as well as the wing.     

Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF beta-receptor by the receptor-like protein tyrosine phosphatase DEP-1

Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF beta-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at -4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position -1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF beta-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions -1 and +3.     

CD98hc (SLC3A2) interaction with the integrin beta subunit cytoplasmic domain mediates adhesive signaling

In mammals, beta1 integrin adhesion receptors generate signals that mediate cell spreading, migration, proliferation, and survival. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin beta subunit cytoplasmic domains (tails) via its heavy chain, CD98hc (SLC3A2), and loss of CD98hc impairs integrin signaling. Here we have used the lack of CD98hc interaction with the Drosophila integrin betaPS tail for a homology scanning analysis that implicated the C-terminal 8 residues of beta3 (Thr(755)-Thr(802)) in CD98hc binding. We then identified point mutations in the beta3 C terminus (T755K and T758M) that abolish CD98hc association and a double mutation in the corresponding residues in the betaPS tail (K839T,M842T), which resulted in gain of CD98hc interaction. Furthermore, the loss of function beta3(T755K) mutation or the gain of function beta3/betaPS(K839T,M842T) led to a loss or gain of integrin-mediated cell spreading, respectively. Thus, we have identified critical integrin residues required for CD98hc interaction and in doing so have shown that CD98c interaction with the integrin beta tail is required for its ability to mediate integrin signaling. These studies also provide new insights into how CD98hc may cooperate with other cytoplasmic domain binding proteins to modulate integrin functions and into the evolution of integrin signaling.     

Differences in regulation of Drosophila and vertebrate integrin affinity by talin

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila alphaPS2betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2betaPS with those of human alphaIIbbeta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2betaPS with those of alphaIIbbeta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIbbeta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2betaPS affinity because of structural features inherent in the alphaPS2betaPS extracellular and/or transmembrane domains.     

Cell-cell adhesion via the ECM: integrin genetics in fly and worm.

Integrins are essential for the development of the two genetically tractable invertebrate model organisms, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Just two integrins are present in C. elegans: one putative RGD binding integrin alphapat-2betapat-3, corresponding to Drosophila alphaPS2betaPS and vertebrate alpha5beta1, alphaVbeta1 and alpha8beta1, and one putative laminin binding integrin alphaina-1betapat-3, corresponding to Drosophila alphaPS1betaPS and vertebrate alpha3beta1, alpha6beta1 and alpha7beta1. In this review, the function of this minimal set of integrins during the development of these two invertebrates is compared. Despite the differences in bodyplan and developmental strategy, integrin adhesion to the extracellular matrix is required for similar processes: the formation of the link that translates muscle contraction into movement of the exoskeleton, cell migration, and morphogenetic interactions between epithelia. Other integrin functions, such as regulation of gene expression, have not yet been experimentally demonstrated in both organisms. Additional proteins have been characterised in each organism that are essential for integrin function, including extracellular matrix ligands and intracellular interacting proteins, but so far different proteins have been found in the two organisms. This in part represents the fact that the characterisation of the full set of interacting proteins is not complete in either system. However, in other cases different proteins appear to be used for similar functions in the two animals. The continued use of genetic approaches to identify proteins required for integrin function in these two model organisms should lead to the identification of the minimal set of conserved components that form integrin adhesive structures.     

An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification.

The backbone modification amide-3, in which -CH2-NH-CO-CH2- replaces -C5'H2-O5'-PO2-O3'-, is studied in the duplex d(G1-C2-G3-T4.T5-G6-C7-G8)*mr(C9-G10-C11-A12-A13-C14-G15+ ++-C16) where . indicates the backbone modification and mr indicates the 2'-OMe RNA strand. The majority of the exchangeable and non-exchangeable resonances have been assigned. The assignment procedure differs from standard methods. The methyl substituent of the 2'-OMe position of the RNA strand can be used as a tool in the interpretation. The duplex structure is a right-handed double helix. The sugar conformations of the 2'-OMe RNA strand are predominantly N-type and the 2'-OMe is positioned at the surface of the minor groove. In the complementary strand, only the sugar of residue T4 is found exclusively in N-type conformation. The incorporation of the amide modification does not effect very strongly the duplex structure. All bases are involved in Watson-Crick base pairs.     

Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.