We have determined the crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition.

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

(Mea-Pro-Leu-Gl~Leu-DpaAla-Arg-NIH?) has been synthasiscd as a lluorogcnic substrate for the matrix mctalloprotcinascs. The highly fluorescent 7.methoxycoumarin group is eflicicntly quenched by energy transfer to the 2,4-dinitrophcnyl group. The punctuated mctalloprotcinase (PUMP, EC 3.42423) cleaves the substrute al the Gly-Leu bond wilh a 1904old increase in fluorrsccncc (A,, 328 nm. A,,, 393 nm). In assays or the buman malrix mclalloprolcinases.Mea-Pro-Lcu.Gly-Lcu.Dpa-Ala_Ar&-NH, is about SO LO 100 timcs more srnsitive than dinitrophcnyl-Pro-leu-Gly-lxu-Trp-Ala-o-Ark-NH2 and continuous assays can be made at enzyme concentrations compardblc IO those used with mucromolccular substrates. Specilicity constiants (k,,lK,) are reported for both synthetic substrates with PUMP. collagenuse. stromelysin and 72 kDa gclatinnsc.

We have previously assigned an integrin ␣ 2 ␤ 1 -recognition site in collagen I to the sequence, GFOGERGVEG-POGPA (O ‫؍‬ Hyp), corresponding to residues 502-516 of the ␣ 1 (I) chain and located in the fragment ␣ 1 (I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports ␣ 2 ␤ 1 -dependent cell adhesion and exhibits divalent cation-dependent binding of isolated ␣ 2 ␤ 1 and recombinant ␣ 2 A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin ␣ 1 A-domain and supports integrin ␣ 1 ␤ 1 -mediated cell adhesion. Triple-helical GFOGER completely inhibits ␣ 2 A-domain binding to collagens I and IV and ␣ 2 ␤ 1 -dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits ␣ 1 A-domain binding to collagen I and strongly inhibits ␣ 1 ␤ 1 -mediated adhesion of Rugli cells to this collagen but has little effect on either ␣1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for ␣ 2 ␤ 1 and in collagen I for ␣ 1 ␤ 1 . Other high-affinity sites in collagen IV mediate its recognition of ␣ 1 ␤ 1 .The integrins are important receptors mediating both cellcell contact and cellular recognition of the extracellular matrix. They are heterodimers comprising an ␣ and a ␤ chain and are classified according to the identity of the latter (1). Integrin recognition sequences have been identified in a number of matrix proteins. RGDX 1 (where X is one of several possible amino acids) occurs in a wide variety of adhesive glycoproteins and recognizes several of the integrins. In fibronectin, for example, RGD recognizes a number of integrins, including ␣ 5 ␤ 1 , ␣ V ␤ 3 , and ␣ IIb ␤ 3 (2, 3).Integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 are the major integrin collagen receptors (4 -6). Each recognizes a variety of collagens, including collagen I, the most abundant and widely distributed of all the collagens. Recognition of collagen IV by integrin ␣ 1 ␤ 1 has been reported to involve an aspartyl residue at position 461 in the ␣ 1 (IV) collagen chain and an arginyl residue at the same residue position in the ␣ 2 (IV) chain (7).Integrin ␣ 2 ␤ 1 plays an essential role in platelet adhesion to collagens in the blood vessel wall under flow conditions (8). This adhesion depends on collagen being in the triple-helical conformation (9) and is important in hemostasis, but it may also be a crucial initiator of thrombosis. Fragmentation of collagen I has indicated the presence of several ␣ 2 ␤ 1 -binding sites throughout the molecule recognized by platelets (9). In particular, fragment ␣ 1 (I)CB3 2 is as good as the parent collagen in supporting ...

TNF-K K converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-K K to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-K K secretion is involved.z 1998 Federation of European Biochemical Societies.

No abstract

Root-associated microbes can improve plant growth, and they offer the potential to increase crop resilience to future drought. Although our understanding of the complex feedbacks between plant and microbial responses to drought is advancing, most of our knowledge comes from non-crop plants in controlled experiments. We propose that future research efforts should attempt to quantify relationships between plant and microbial traits, explicitly focus on food crops, and include longer-term experiments under field conditions. Overall, we highlight the need for improved mechanistic understanding of the complex feedbacks between plants and microbes during, and particularly after, drought. This requires integrating ecology with plant, microbiome, and molecular approaches and is central to making crop production more resilient to our future climate.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described`metallosheddase' cleavage sites of tumour necrosis factor K K, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.z 2000 Federation of European Biochemical Societies.