Elsa D. Garcin

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg1400, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low levels, and a defensive cytotoxin at higher levels. The high active-site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock, and cancer. Our structural and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a novel specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents towards remote specificity pockets, accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active-site conservation.

DNA apurinic-apyrimidinic site binding and excision by endonuclease IV.

Escherichia coli endonuclease IV is an archetype for an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair. Here biochemical, mutational and crystallographic characterizations reveal a three–metal ion mechanism for damage binding and incision. The 1.10-Å resolution DNA-free and the 2.45-Å resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. The 1.45-Å resolution DNA-product complex structure shows how Tyr72 caps the active site, tunes its dielectric environment and promotes catalysis by Glu261-activated hydroxide, bound to two Zn2+ ions throughout catalysis. These structural, mutagenesis and biochemical results suggest general requirements for abasic site removal in contrast to features specific to the distinct endonuclease IV α-β triose phosphate isomerase (TIM) barrel and APE1 four-layer α-β folds of the apurinic-apyrimidinic endonuclease families.

Hydrogenase: A hydrogen-metabolizing enzyme. What do the crystal structures tell us about its mode of action?

Hydrogenases are proteins which metabolize the most simple of chemical compounds, molecular hydrogen, according to the reaction H2<-->2H+ + 2e-. These enzymes are found in many microorganisms of great biotechnological interest such as methanogenic, acetogenic, nitrogen fixing, photosynthetic or sulfate-reducing bacteria. The X-ray structure of a dimeric [NiFe] hydrogenase together with a wealth of biophysical, biochemical and genetic studies have revealed that the large subunit contains the bimetallic [Ni-Fe] active site, with biologically uncommon CO and CN ligands to the iron, whereas the small subunit contains three iron-sulfur cluster. During catalysis, the nickel atom is most likely responsible for a base-assisted heterolytic cleavage of the hydrogen molecule whereas the iron atom could be redox active. Specific channels are probably required for the transfer of the chemical reaction partners (H2, H+ and e-) between the active site, deeply buried inside the protein, and the molecular surface. The generation of a functional enzyme, including the assembly of the complex catalytic center, requires maturation and involves a large number of auxiliary proteins which have been partly characterized by molecular biology.

C-terminal tail residue Arg1400 enables NADPH to regulate electron transfer in neuronal nitric-oxide synthase

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg1400 in the C-terminal tail regulatory element and the 2′-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg1400 in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent Km for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg1400-2′-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.

Interfacial residues promote an optimal alignment of the catalytic center in human soluble guanylate cyclase: heterodimerization is required but not sufficient for activity.

Soluble guanylate cyclase (sGC) plays a central role in the cardiovascular system and is a drug target for the treatment of pulmonary hypertension. While the three-dimensional structure of sGC is unknown, studies suggest that binding of the regulatory domain to the catalytic domain maintains sGC in an autoinhibited basal state. The activation signal, binding of NO to heme, is thought to be transmitted via the regulatory and dimerization domains to the cyclase domain and unleashes the full catalytic potential of sGC. Consequently, isolated catalytic domains should show catalytic turnover comparable to that of activated sGC. Using X-ray crystallography, activity measurements, and native mass spectrometry, we show unambiguously that human isolated catalytic domains are much less active than basal sGC, while still forming heterodimers. We identified key structural elements regulating the dimer interface and propose a novel role for residues located in an interfacial flap and a hydrogen bond network as key modulators of the orientation of the catalytic subunits. We demonstrate that even in the absence of the regulatory domain, additional sGC domains are required to guide the appropriate conformation of the catalytic subunits associated with high activity. Our data support a novel regulatory mechanism whereby sGC activity is tuned by distinct domain interactions that either promote or inhibit catalytic activity. These results further our understanding of heterodimerization and activation of sGC and open additional drug discovery routes for targeting the NO–sGC–cGMP pathway via the design of small molecules that promote a productive conformation of the catalytic subunits or disrupt inhibitory domain interactions.

YC-1 binding to the β subunit of soluble guanylyl cyclase overcomes allosteric inhibition by the α subunit.

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the β subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 μM, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 μM. Pfizer compound 25 bound ∼10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca β H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 μM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

A dimer interface mutation in glyceraldehyde 3-phosphate dehydrogenase regulates its binding to AU-rich RNA.

DOI 10.1074/jbc.A114.618165 A dimer interface mutation in glyceraldehyde 3-phosphate dehydrogenase regulates its binding to AU-rich RNA. Michael R. White, Mohd M. Khan, Daniel Deredge, Christina R. Ross, Royston Quintyn, Beth E. Zucconi, Vicki H. Wysocki, Patrick L. Wintrode, Gerald M. Wilson, and Elsa D. Garcin PAGE 1780: The charge state distributions shown for the mass spectra peaks in Fig. 7B were not correct. The correct charge state distributions are shown in the revised Fig. 7B. This correction does not affect the results or conclusions of this work. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 7, p. 4129, February 13, 2015

Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer.

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys842) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys842. Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys842 is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.

The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein.

The glycolytic protein, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), has a vast array of extraglycolytic cellular functions, including interactions with nucleic acids. GAPDH has been implicated in the translocation of transfer RNA (tRNA), the regulation of cellular messenger RNA (mRNA) stability and translation, as well as the regulation of replication and gene expression of many single‐stranded RNA viruses. A growing body of evidence supports GAPDH–RNA interactions serving as part of a larger coordination between intermediary metabolism and RNA biogenesis. Despite the established role of GAPDH in nucleic acid regulation, it is still unclear how and where GAPDH binds to its RNA targets, highlighted by the absence of any conserved RNA‐binding sequences. This review will summarize our current understanding of GAPDH‐mediated regulation of RNA function. WIREs RNA 2016, 7:53–70. doi: 10.1002/wrna.1315