The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782–785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray “closed” conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally-derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

Advanced NMR and theoretical studies reveal detailed insights into monomeric and dimeric structures plus deviating experimental and theoretical E/Z-populations.

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of fulllength matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs. Matrix metalloproteinases (MMP)2 are extracellular hydrolytic enzymes involved in a variety of processes including connective tissue cleavage and remodeling (1-3). All 23 members of the family are able to cleave simple peptides derived from connective tissue components such as collagen, gelatin, elastin, etc. A subset of MMPs is able to hydrolyze more resistant polymeric substrates, such as cross-linked elastin, and partially degraded collagen forms, such as gelatin and type IV collagens (4). Intact triple helical type I-III collagen is only attacked by collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14 (5-12). Although the detailed mechanism of cleavage of single chain peptides by MMP has been largely elucidated (13-19), little is known about the process of hydrolysis of triple helical collagen. In fact, triple helical collagen cannot be accommodated in the substrate-binding groove of the catalytic site of MMPs (9).All MMPs (but MMP-7) in their active form are constituted by a catalytic domain (CAT) and a hemopexin-like domain (HPX) (20 -22). The CAT domain contains two zinc ions and one to three calcium ions. One zinc ion is at the catalytic site and is responsible for the activity, whereas the other metal ions have structural roles. The isolated CAT domains retain full catalytic activity toward simple peptides and single chain polymeric substrates such as elastin, whereas hydrolysis of triple helical collagen also requires the presence of the HPX domain (9, 23-25). It has been shown that the isolated CAT domain regains a small fraction of the activity of the full-length (FL) protein when high amounts of either inactivated full-length proteins or isolated HPX domains are added to the assay solution (9). Finally, it has been shown that the presence of the HPX domain alone alters the CD spectrum of triple helical collagen in a way that suggests its partial unwinding (26,27). It is tempting to speculate that full-length collagenases attack collagen by first locally unwinding the triple helical structure with the help of the HPX domain and then cleaving the resulting, exposed, single filaments (9, 28).Until 2007, three-dimensional structures of full-length MMPs had been reported only for collagenase MMP-1 (29 -31) and gelatinase MMP-2 (32). The structures of the two proteins are very similar and show a compact arrangement of the two ...

Matrix metalloproteinases (MMP) are an important family of 23 proteins which are involved in a number of extracellular processes including the degradation of the extracellular matrix.1,2 All active MMPs but MMP-7 are constituted by two domains, a catalytic (CAT) and a hemopexin-like (HPX) domain. The CAT and HPX domains are connected by a linker whose length varies from 14 to 68 AA. 3,4 For the majority of MMPs the linker is relatively short (14-23 AA) while for MMP-9 and MMP-15, at the other extreme, the intervening residues between the CAT and HPX domains (63 and 68 AA, respectively), constitute a further, highly glycosylated, domain termed OG domain. The CAT domain alone bears full proteolytic activity towards a range of peptides and proteins. However, efficient proteolysis of, for instance, triple helical collagen requires the full-length active protein. For this reason it is often hypothesized that the HPX domain helps the local unwinding of the triple helix, in such a way that a single peptide strand can be accommodated in the active site of the CAT domain and cleaved. It has been also hypothesized that a relative mobility of the HPX domain is necessary for this function. In case of full-length MMP-12 the low resolution crystal structure we collected (Figure 1) was largely sufficient to establish that the structure is less compact, and the relative orientation of the two domains totally different, with respect to the four Xray structures of MMP-1 and MMP-2 already described. Moreover NMR data collected in solution show that the two domains are not held rigidly to one another, but must undergo independent motions. Therefore SAXS analysis has been performed in order to investigate the types and variety of the sampled conformations. Synchrotron X-ray scattering data from FL-MMP-12 solutions was collected on the X33 beamline, of the EMBL (DESY, Hamburg), using a MAR345 image plate detector. The scattering patterns were measured with a 2 minute exposure time for several solute concentrations in the range from 0.8 to 8.3 mg/ml. The processed X-ray scattering pattern from FL-MMP-12 presented in Figure 1 yields a molar mass 369

NMR H-bond analysis reveals an offset of internal and external acidities of catalysts and allows for a detailed reactivity analysis.

No abstract

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/β-pregnane-3,20-dione or 5β-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/β-pregnane-3,20-dione, among which the 5β-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 μM versus 1.2 and 10.6 μM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5β-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 μM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

Dedicated to the Centenary of the Italian Chemical Society MMP-12 (matrix metalloproteinase 12)is an important protein of the MMP family. [1][2][3][4] Its substrate is elastin, [5][6][7][8] which is composed of a cross-linked insoluble network of polypeptide chains of tropoelastin of MW 65 kDa each. [9] Insoluble elastin is responsible for keeping some extracellular connective tissues elastic.The full-length MMP-12 is made up by a catalytic (CAT) domain and a hemopexin-like (HPX) domain. The two domains have relatively large conformational freedom, [10] as also recently found in MMP-1 [11] and MMP-9. [12] The role of the HPX domain is not certain, as the CAT domain is always active by itself, [13] even if it may have a smaller turnover for some substrates. [14] Experimental structures and models, representative of the binding mode of peptide substrates on protein active site are already present in the Protein Data Bank. However, the interaction of native elastin with full length MMP-12 and its [a] Prof.