Marco Fragai

Evidence of reciprocal reorientation of the catalytic and hemopexin-like domains of full-length MMP-12

The proteolytic activity of matrix metalloproteinases toward extracellular matrix components (ECM), cytokines, chemokines, and membrane receptors is crucial for several homeostatic and pathological processes. Active MMPs are a family of single-chain enzymes (23 family members in the human genome), most of which constituted by a catalytic domain and by a hemopexin-like domain connected by a linker. The X-ray structures of MMP-1 and MMP-2 suggest a conserved and well-defined spatial relationship between the two domains. Here we present structural data for MMP-12, suitably stabilized against self-hydrolysis, both in solution (NMR and SAXS) and in the solid state (X-ray), showing that the hemopexin-like and the catalytic domains experience conformational freedom with respect to each other on a time scale shorter than 10(-8) s. Hints on the probable conformations are also obtained. This experimental finding opens new perspectives for the often hypothesized active role of the hemopexin-like domain in the enzymatic activity of MMPs.

Interdomain Flexibility in Full-length Matrix Metalloproteinase-1 (MMP-1)

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length 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.

NMR-based techniques in the hit identification and optimisation processes

In this review, the use of general NMR spectroscopy techniques to detect ligand binding and to monitor enzyme kinetics and inhibition, which appear particularly useful in hit identification and validation, is reiterated. Furthermore, the use of NMR-based strategies for lead optimisations that are based on either iterative derivatisations of an initial core structure or on linking fragments that occupy adjacent pockets in the target’s binding site will also be described. Several recent examples will be reported and the use of these techniques in cases when the three dimensional structure of the target protein is known will be discussed.

Structural basis of serine/threonine phosphatase inhibition by the archetypal small molecules cantharidin and norcantharidin

The inhibition of a subgroup of human serine/threonine protein phosphatases is responsible for the cytotoxicity of cantharidin and norcantharidin against tumor cells. It is shown that the anhydride rings of cantharidin and norcantharidin are hydrolyzed when bound to the catalytic domain of the human serine/threonine protein phosphatases 5 (PP5c), and the high-resolution crystal structures of PP5c complexed with the corresponding dicarboxylic acid derivatives of the two molecules are reported. Norcantharidin shows a unique binding conformation with the catalytically active Mn2PP5c, while cantharidin is characterized by a double conformation in its binding mode to the protein. Different binding modes of norcantharidin are observed depending of whether the starting ligand is in the anhydride or in the dicarboxylic acid form. All these structures will provide the basis for the rational design of new cantharidin-based drugs.

Entropic contribution to the linking coefficient in fragment based drug design: a case study.

For several drug leads obtained by tethering weak binding ligands, the dissociation constant is smaller than the product of those of the individual fragments by a factor named the linking coefficient, E. This favorable contribution is attributed to the entropic gain that is realized when two weak binding ligands are tethered. Here we show a case study where the linking coefficient is strikingly small (E = 2.1 x 10(-3) M(-1)) and its totally entropic nature is demonstrated.

Persistent contrast enhancement by sterically stabilized paramagnetic liposomes in murine melanoma

In the present research, we investigated the use of paramagnetic liposomes as contrast agents (CAs) for the detection of solid tumors. The liposomes were sterically stabilized by a polyethylene glycol (PEG) coating, and their size was constrained to ∼100 nm. Dimyristoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐diethylene‐triaminepentaacetate (DMPE‐DTPA) was used as the gadolinium‐carrying fatty acid chain. The relaxation properties were characterized through nuclear magnetic relaxation dispersion (NMRD) measurements, and analyzed with the use of theories and computer programs that are adequate for slowly rotating systems. Their relaxivity at 1.5 T was found to be acceptable for in vivo use. We then tested the liposomes against B16‐F10 murine melanomas using standard T1‐weighted schemes at 1.5 T, and concentrations corresponding to 0.03 mmol/kg of gadolinium (i.e., three to six times lower than the concentration of the small gadolinium complexes in clinical use). The blood half‐life was found to be 120 ± 20 min. The experiments show a good contrast enhancement in the tumor (33% ± 22%) 2 hr after administration, a further increase (43 ± 27%) 20 hr after administration, and a decrease (25% ± 14%) 54 hr after administration. High persistence of the CA was also observed in the liver and intestine, as expected in a hepatobiliar excretion pathway. Magn Reson Med 52:669–672, 2004.

Sulfonamide‐Functionalized Gadolinium DTPA Complexes as Possible Contrast Agents for MRI: A Relaxometric Investigation

A novel Gd-DTPA derivative with a built-in sulfonamide (SA) was synthesized as a contrast agent for MRI. The complex was designed to selectively target the enzyme carbonic anhydrase. It is shown that the longitudinal relaxation rates of aqueous solutions of Gd-DTPA-SA in the presence of carbonic anhydrase increase significantly. The binding constant is determined to be 15,000 ± 5,000 M–1. This value ensures substantial formation of the carbonic anhydrase adduct at imaging concentrations of Gd-DTPA-SA. The complex interacts with erythrocytes, presumably due to a high affinity for the carbonic anhydrase present on the outer surface of the latter. This takes place even though the enzyme has a low abundance and is easily saturated by small amounts of Gd-DTPA-SA. The interaction of Gd-DTPA-SA with serum proteins is negligibly small. Therefore, the complex could potentially be tested as a selective contrast agent for compartments outside the blood pool.

Examination of Matrix Metalloproteinase-1 in Solution: A PREFERENCE FOR THE PRE-COLLAGENOLYSIS STATE*

Background: Matrix metalloproteinase-1 (MMP-1) collagenolysis relies on interdomain flexibility. Results: In all high maximum occurrence conformations, the MMP-1 hemopexin-like domain residues reported responsible for binding to the collagen triple-helix are solvent exposed. Conclusion: MMP-1 in solution is poised to interact with collagen and proceed along the steps of collagenolysis. Significance: The maximum occurrence approach can evaluate the predominant domain conformations for numerous multidomain enzymes. Catalysis of collagen degradation by matrix metalloproteinase 1 (MMP-1) has been proposed to critically rely on flexibility between the catalytic (CAT) and hemopexin-like (HPX) domains. A rigorous assessment of the most readily accessed conformations in solution is required to explain the onset of substrate recognition and collagenolysis. The present study utilized paramagnetic NMR spectroscopy and small angle x-ray scattering (SAXS) to calculate the maximum occurrence (MO) of MMP-1 conformations. The MMP-1 conformations with large MO values (up to 47%) are restricted into a relatively small conformational region. All conformations with high MO values differ largely from the closed MMP-1 structures obtained by x-ray crystallography. The MO of the latter is ∼20%, which represents the upper limit for the presence of this conformation in the ensemble sampled by the protein in solution. In all the high MO conformations, the CAT and HPX domains are not in tight contact, and the residues of the HPX domain reported to be responsible for the binding to the collagen triple-helix are solvent exposed. Thus, overall analysis of the highest MO conformations indicated that MMP-1 in solution was poised to interact with collagen and then could readily proceed along the steps of collagenolysis.

Unraveling hidden regulatory sites in structurally homologous metalloproteases

Monitoring enzymatic activity in vivo of individual homologous enzymes such as the matrix metalloproteinases (MMPs) by antagonist molecules is highly desired for defining physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous enzymes often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Yet, the challenge remains to identify such regulatory alternative sites that are often hidden and scattered over different locations on the proteins surface. We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers share the same chemical nature, providing versatile nonspecific binding reactivity that allows to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. This approach includes diverse experimental analysis, such as structural spectroscopic analyses, NMR, and protein crystallography combined with computational prediction of effector binding sites. We demonstrate how our approach works by analyzing members of the MMP family that possess a unique set of such sites. Our work provides a proof of principle for using ligand effectors to unravel hidden regulatory sites specific to members of the structurally homologous MMP family. This approach may be exploited for the design of novel molecular effectors and therapeutic agents affecting protein catalytic function via interactions with structure-specific regulatory sites.

Solid-State NMR of Matrix Metalloproteinase 12: An Approach Complementary to Solution NMR

Solid-state NMR (SS NMR) is a technique that has shown a rapid development in recent years. The exciting progress in sample-preparation methods, tailored pulse sequences, and instrumentation now make the investigation of relatively large proteins possible. In spite of these developments, the number of proteins for which an almost complete solid-state assignment is available is still small. Interestingly, it is generally observed that C chemical shifts do not change much on passing from solution to microcrystalline samples (the differences generally being <1 ppm). This opens the way to a fast liquid-based solid-state assignment, in which the available liquid assignment is transferred to the solid-state spectra, and only a minimal number of solid-state spectra are acquired. The possibility of obtaining a solid-state assignment in a short time is valuable, as it will permit, for instance, the investigation of a protein as part of larger aggregates (oligomerization, protein– protein complexes) without the line broadening due to the increase in molecular weight observed with solution NMR. In this work we demonstrate that it is possible to provide a large fraction of the solid-state NMR assignment of a relatively large protein (17 kDa) rapidly by using a pair of experiments (CP MAS proton-driven spin diffusion (PDSD) and J-decoupled PDSD). These spectra can be acquired in a limited amount of time (12–15 h each), and manually assigned in a few days by using the available liquid-state assignment as a guideline. 3D NCACX and NCOCX PDSD spectra fully validate the obtained assignment and further increase the overall fraction of assigned peaks. However, they require considerably more experimental time. For this investigation we selected a microcrystalline sample of the catalytically active domain of the zinc-containing matrix metalloproteinase 12 (Zn-MMP-12, 159 AA, 17.6 kDa), for which one X-ray structure and the solution NMR assignment are available. The crystallographic structure indicates that the catalytically active domain is composed of three a-helices (44 AA, 28% of the total residues) and seven b-strands (27 AA, 17%). The remaining 88 residues do not form regular secondary structure. The protein contains two zinc(II) ions and three calcium(II) ions. One of the zinc ions is responsible for the catalytic activity. The SS NMR sample was prepared by crystallizing the protein from 30% poly(ethylene glycol) (PEG) 8000 according the published procedure. The microcrystalline precipitate began to appear after 12 h, and the crystallization was complete after 1–2 days. Figure 1 shows the C,C CP MAS PDSD spectrum acquired at 16.4 T (700 MHz H Larmor frequency) and a MAS frequency wR/2p=11.5 kHz, with a mixing time of 15 ms. The assignment