Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl ‘decoy receptor’ that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high affinity Axl variant caused structural alterations in side chains across the Gas6/Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc-fusion, the engineered decoy receptor bound to Gas6 with femtomolar affinity, an 80-fold improvement compared to the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.assembly line enzymes | polyketide synthase | protein-protein interactions M odular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) are assembly lines of catalytic modules responsible for the biosynthesis of polyketide natural products (1-6). A fundamental challenge toward elucidating their molecular logic is to understand the mechanism by which covalently bound biosynthetic intermediates are sequentially accessed by individual catalytic sites of the synthase. Most notably, each module of a polyketide synthase (PKS) has an acyl carrier protein (ACP) domain that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze polyketide chain elongation, and subsequently engages with the KS domain of the next module to facilitate forward chain transfer (Fig. 1). The simplest explanation for the orderly progress of a growing polyketide chain along a multimodular PKS is that each ACP is equivalently recognized by both KS partners, and that selection of the appropriate KS-ACP pair at a given point in the catalytic cycle is solely dictated by the identity of the substrate anchored on the ACP. (In the chain elongation reaction, a nucleophilic malonyl extender unit is attached to the ACP, whereas the growing polyketide chain itself is attached to the ACP via an electrophilic thioester linkage when it is ready for forward transfer.) Previous studies, however, suggest that protein-protein recognition between the KS and the ACP domains also plays an important role in both reactions (7-10). Moreover, this protein-protein recognition not only influences the specificity (k cat ∕K M ) of each reaction, but also the maximum rate constant (k cat ).In the course of our efforts to elucidate the principles that govern KS-ACP recognition during chain elongation and chain transfer, we have made two unexpected and potentially important observations. First, notwithstanding the fact that the same active sites are deployed in both reactions, the structural elements that mediate KS-ACP recognition are entir...

Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.A fundamental challenge to our understanding of multimodular polyketide synthases (PKSs) is the ability to explain the unidirectional translocation of growing polyketide chains through these enzymatic assembly lines. The 6-deoxyerythronolide B synthase (DEBS; Fig. 1) is arguably the most well studied example of assembly line PKSs (1-4). Each module of DEBS has an acyl carrier protein (ACP) that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze a single round of polyketide chain elongation ( Fig. 2). At this point, the ACP-bound intermediate is precluded from back-transfer to the same KS domain and is instead translocated to the KS domain of the downstream module (Fig. 2).In the course of our investigations into the mechanism of intermodular chain translocation (Fig. 2, reaction 1) and intramodular chain elongation (Fig. 2, reaction 4) within DEBS, we discovered that the specificity of these two reactions is controlled by protein-protein interfaces involving distinct regions of the ACP domain (5). In the present study, we have used site-directed mutagenesis to identify ACP residues that contribute to the observed specificity. In turn, these residue-level constraints were exploited to validate the proposed structural model for ACP docking during chain elongation (5), as well as to develop an analogous in silico model for chain translocation. Generalization of these models to three naturally occurring PKSs has revealed a programming pattern that establishes a ratchet mechanism that accurately explains the unique chain translocation pathway of each PKS. As a test of this ratcheting mechanism in PKS assembly lines, we engineered a module of DEBS to iteratively catalyze two successive rounds of chain elongation instead of only one. Results and DiscussionIdentification of ACP Residues That Contribute to Chain Elongation Specificity. All ACPs from fatty acid and polyketide synthases are all-helical bundles comprised of three major α-helices connected by two structured loops (Fig. 3A). In earlier work (5) we showe...

named as inventors. A.J. Giaccia and A.C. Koong are cofounders of Ruga Corp., a company that has licensed this patent.

The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF 3 -S-ACP, was developed as a 19 F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.