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Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure-activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

SUMMARY Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions., Graphical Abstract, In Brief Yan et al. describe the structure of neurofibromin (GAP-related domain) in complex with SPRED1(EVH1) and KRAS and suggest a mechanism by which this interaction is regulated during normal cell signaling and in cells with activated receptor tyrosine kinase oncogenes. The neurofibromin-SPRED1 interaction interface provides a rationale for mutations observed in Legius syndrome.

International audience; Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intra-molecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 doesn’t dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 towards the CTD peptide by four fold. To our surprise, even though the intra-molecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription.

The biosynthesis of the C ring of the anti-tumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from L-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from the one processed by 4-OT. In order to establish the function and mechanism of TomN and its relationship to 4-OT, kinetic, mutagenic, and structural studies have been carried out. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutant of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with an RMS deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.

Phosphagen kinase (PK) family members catalyze the reversible phosphoryl transfer between phosphagen and ADP to reserve or release energy in cell energy metabolism. The structures of classic quaternary complexes of dimeric creatine kinase (CK) revealed asymmetric ligand binding states of two protomers, but the significance and mechanism remain unclear. To understand this negative cooperativity further, we determined the first structure of dimeric arginine kinase (dAK), another PK family member, at 1.75 A, as well as the structure of its ternary complex with AMPPNP and arginine. Further structural analysis shows that the ligand-free protomer in a ligand-bound dimer opens more widely than the protomers in a ligand-free dimer, which leads to three different states of a dAK protomer. The unexpected allostery of the ligand-free protomer in a ligand-bound dimer should be relayed from the ligand-binding-induced allostery of its adjacent protomer. Mutations that weaken the interprotomer connections dramatically reduced the catalytic activities of dAK, indicating the importance of the allosteric propagation mediated by the homodimer interface. These results suggest a reciprocating mechanism of dimeric PK, which is shared by other ATP related oligomeric enzymes, e.g., ATP synthase.