SUMO E2/E3 Enzyme Co-Recognize Substrates and Confers High Substrates Specificity

Although various technologies can determine protein-protein interaction affinity, Kd, current approaches require at least one interacting protein to be purified. Here, we present a development of high-throughput approach to determine protein interaction affinity for un-purified interacting proteins using quantitative FRET assay. We developed this approach first using SUMO E2 conjugating enzyme, Ubc9, and SUMO substrate, RanGap1c. The interaction affinities from two purified proteins and two un-purified proteins in the presence of BSA, bacterial extracts or two mixtures are all in excellent agreement with that obtained from the SPR measurement. We then applied this approach to determine the interaction affinities of SUMO E3 PIAS1 and Ubc9 with a SUMO substrate, influenza virus protein, NS1, and, for the first time, the SUMO E3 ligase-substrate interaction affinity is determined, which enables us to provide a kinetics explanation for the two-enzyme substrate recognition mode. In general, our studies allow high-throughput determination of protein interacting affinity without any purification, and both the approach and scientific discoveries can be applied to proteins that are difficult to be expressed in general.

Conformational flexibility of EptA driven by an interdomain helix provides insights for enzyme–substrate recognition

Many pathogenic gram-negative bacteria have developed mechanisms to increase resistance to cationic antimicrobial peptides by modifying the lipid A moiety. One modification is the addition of phosphoethanolamine to lipid A by the enzyme phosphoethanolamine transferase (EptA). Previously we reported the structure of EptA from Neisseria, revealing a two-domain architecture consisting of a periplasmic facing soluble domain and a transmembrane domain, linked together by a bridging helix. Here, the conformational flexibility of EptA in different detergent environments is probed by solution scattering and intrinsic fluorescence-quenching studies. The solution scattering studies reveal the enzyme in a more compact state with the two domains positioned close together in an n-dodecyl-β-D-maltoside micelle environment and an open extended structure in an n-dodecyl-phosphocholine micelle environment. Intrinsic fluorescence quenching studies localize the domain movements to the bridging helix. These results provide important insights into substrate binding and the molecular mechanism of endotoxin modification by EptA.

Extracellular endosulfatase Sulf-2 harbours a chondroitin/dermatan sulfate chain that modulates its enzyme activity

Sulfs represent a class of unconventional sulfatases, which differ from all other members of the sulfatase family by their structures, catalytic features and biological functions. Through their specific endosulfatase activity in extracellular milieu, Sulfs provide an original post-synthetic regulatory mechanism for heparan sulfate complex polysaccharides and have been involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) features a unique polysaccharide post-translational modification. We identified a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate binding domain. We found that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo using a mouse model of tumorigenesis and metastasis. In addition, we showed that mammalian hyaluronidase acted as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a unique proteoglycan enzyme and its newly-identified GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute in clarifying the conflicting data on the activities of the Sulfs and introduce a new paradigm into the study of these enzymes.