Introduction 1 , 2 3 4 5 , 6 7,9 8 , 10 11 12 13 The three-dimensional structure of the human MMP-1 (E200A), an active site mutant, reported here takes us a step closer towards a more complete understanding of the interaction of the collagenases with the triple-helical collagens. It reveals new features of the active protease and provides a platform for understanding the structural changes that accompany zymogen activation. Results Overall structure P 2 Table 1 Figure 1 13 14 α Table 2 Catalytic domain Figure 2 15 15 16 , 17 Figure 3 18 13 Figure 3 Table 3 Table 3 Linker region 19 13 Figure 4 Table 4 Table 4 Figure 4 Figure 4 Hemopexin-like domain Table 3 12 Comparison with the porcine MMP-1 and the human proMMP-1 structures 13 12 α Figure 5 Table 5 Figure 5 Table 5 et al. 13 cis Discussion versus Figure 3 17 20 Figure 2 via 19 , 21 Figure 4 21 19 22 Figure 5 Figure 5 23 Implications for collagenolysis et al. 13 24 cis via cis 22 The structure of the active form of human MMP-1 has provided us insights on the conformational changes that occur upon activation of the pro-enzyme and these are valuable clues towards understanding the mechanism of collagenolysis. A better understanding of the mechanism of collagen cleavage will assist towards design of inhibitors that would specifically interfere with collagenolysis without affecting (beneficial) the cleavage of other substrates. Experimental Procedures Cloning, protein expression and protein purification 22 22 2 Crystallisation, data collection and processing MMP-1 (E200A) was crystallised using the hanging drop vapour-diffusion method. The protein (2 μl at a concentration of 21 mg/ml) was mixed with 2 μl of the reservoir solution containing 0.1 M Tris (pH 7.5), 1.5 M ammonium formate and 10% (w/v) polyethylene glycol (PEG) 8000. Crystals appeared and grew to their maximum size within two weeks at 16 °C. A cryoprotectant solution prepared by supplementing the reservoir with 25% (v/v) glycerol enabled the crystals to be flash-frozen in liquid nitrogen. 25 P 2 a b c 26 B 2 Table 1 Structure determination 26 13 Refinement 27 R R free 28 26 R cryst R free R cryst R free B 27 F o F c F o F c 29 F o F c R cryst R free 26 Table 1 Protein Data Bank accession codes The atomic coordinates and the structure factors of the human MMP-1 (E200A) have been deposited with the RCSB Protein Data Bank (accession codes 2CLT and R2CLTSF, respectively).