Solid‐State Structural Characterization of Cutinase–ECE‐Pincer–Metal Hybrids

Abstract

The first crystal structures of lipases that have been covalently modified through site-selective inhibition by different organometallic phosphonate-pincer-metal complexes are described. Two ECE-pincer-type d(8)-metal complexes, that is, platinum (1) or palladium (2) with phosphonate esters (ECE = [(EtO)-(O=)P(-O-C(6)H(4)-(NO(2))-4)(-C(3)H(6)-4-(C(6)H(2)-(CH(2)E)(2))](-); E = NMe(2) or SMe) were introduced prior to crystallization and have been shown to bind selectively to the Ser(120) residue in the active site of the lipase cutinase to give cut-1 (platinum) or cut-2 (palladium) hybrids. For all five presented crystal structures, the ECE-pincer-platinum or -palladium head group sticks out of the cutinase molecule and is exposed to the solvent. Depending on the nature of the ECE-pincer-metal head group, the ECE-pincer-platinum and -palladium guests occupy different pockets in the active site of cutinase, with concomitant different stereochemistries on the phosphorous atom for the cut-1 (S(P)) and cut-2 (R(P)) structures. When cut-1 was crystallized under halide-poor conditions, a novel metal-induced dimeric structure was formed between two cutinase-bound pincer-platinum head groups, which are interconnected through a single mu-Cl bridge. This halide-bridged metal dimer shows that coordination chemistry is possible with protein-modified pincer-metal complexes. Furthermore, we could use NCN-pincer-platinum complex 1 as site-selective tool for the phasing of raw protein diffraction data, which shows the potential use of pincer-platinum complex 1 as a heavy-atom derivative in protein crystallography.

Highlights of 45 years of research: A personal account

“…The overlay of X-rays of two MX(ECE)-Cutinase hybrids, i.e. P* n -ECE-MX ¼ (S p )-NCNPtCl and (R p )-SCSPdBr), respectively reveals opposite chirality of the respective phosphorous centers at their inhibition point in the lipase [68] (see Fig. 22).…”

Site‐Selective Ser‐Hydrolase Labelling with a Luminescent Organometallic NCN–Platinum Complex

“…With the organometallic ECE-pincer-metal phosphonate inhibitors [19][20][21] developed by our group, we have been exploiting the special properties of the heavy-metal atom probe as, for example, coordination target for phosphanes and as phasing tool for the elucidation of protein crystal structures. [21,33] Interestingly, many different coordination complexes with exciting luminescent properties are known from the literature, [34][35][36] but only very few of them have been used in the labelling of biomolecules, [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] which is mainly caused by the instability (e.g., water or redox sensitivity) of these complexes under biological conditions.…”

“…By doing so, novel, unnatural features can be added to the protein, which can be exploited in coordination studies involving the metal centre or in the phasing of protein diffraction data. [21] Phosphonates have long been known to be substrate analogues for serine hydrolases, and even phosphonates with various organic fluorescent reporter tags have been studied by different groups. [17,22,[29][30][31] So far, phosphonates substituted by organic dyes [e.g., fluorescent dansyl (Figure 1), [22] rhodamine [15] and p-nitrobenzofurazan [32] groups] have [20,21] of an organic luminescent dansyl phosphonate inhibitor (middle) [22] and of the organometallic luminescent target inhibitor 6 (right).…”

“…Organometallic pincer complexes possess a covalent metal-carbon bond, which is completed by two ortho-chelated metal-heteroatom interactions. [19][20][21]27,28,[57][58][59] This combination of metal-ligand interactions leads to a high stability in aqueous and biological media, which in turn led us to expect that these complexes would be stable luminescence labels for protein profiling.…”

“…[21,33] Interestingly, many different coordination complexes with exciting luminescent properties are known from the literature, [34][35][36] but only very few of them have been used in the labelling of biomolecules, [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] which is mainly caused by the instability (e.g., water or redox sensitivity) of these complexes under biological conditions. Inspired by the high quantum yields of various organoplatinum complexes, as demonstrated by the Williams [25][26][27]55] and other groups [56] and by our own results on the design and applications of novel organometallic phosphonate inhibitors, we embarked on the modification of one of these highly luminescent NCN-platinum complexes [57] (complex 1, Scheme 1) to develop a novel luminescent organometallic NCN-platinum phosphonate inhibitor (6, Figure 1, Scheme 1).…”