Rajnandani Kashyap

Many photosynthetic bacteria rely on the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) to sustain (bacterio)chlorophyll synthesis in the dark. DPOR couples ATP hydrolysis to electron transfer from a reducing component (L-protein) to a catalytic component (NB-protein) that reduces protochlorophyllide. NB-protein is an α ₂ β ₂ heterotetramer, and each heterodimeric half contains an active site and a [Fe ₄ S ₄ ] cluster. Although the two halves are identical, they react sequentially rather than in parallel. How the two halves communicate has remained unknown. Here we show, using Cu K-edge x-ray absorption spectroscopy and cryo-EM, that the NB-protein tetramer interface holds a binuclear, sulfur-bridged [Cu ₂ S ₂ ] cluster. Each Cu is reduced and three-coordinate, with short Cu-S and Cu-Cu distances (2.26 and 2.62 Å). Mutagenesis excludes the nearest cysteine as the bridging ligand, supporting inorganic sulfide as the most likely sulfur donor. To our knowledge, this is the first sulfide-bridged dicopper cluster identified in a protein. The cluster is conserved in three purple non-sulfur bacteria and connects the BchB C-terminal helices that contact opposing active sites. Loss of Cu abolishes DPOR activity and asymmetric L-protein binding without disturbing overall fold. We propose that the cluster relays substrate-binding information between active sites, suggesting a metal-mediated mechanism for cooperativity in nitrogenase-like enzymes.

The insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog-specific functions despite high structural similarity. Each paralog contains six RNA-binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site-specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long-range arrangements of RNA-binding domains and linkers without altering the secondary structure, and with only modest effects on RNA-binding affinity. Critically, pSer-driven rearrangements occur both in the RNA-free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog-specific, phosphorylation-dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post-translational mechanism that tunes IGF2BP paralog dynamics across free and RNA-bound states to program target mRNA selection, processing, and translational fate.

Nitrogenases are metalloenzymes that catalyze the reduction of atmospheric dinitrogen to ammonia, sustaining the global nitrogen cycle. While bacterial nitrogenase has been extensively characterized, the architecture and regulation of archaeal nitrogenases remain unknown despite longstanding evidence of nitrogen fixation in methanogens. Here we report a 3.2 Å cryo-electron microscopy structure of a native nitrogenase–PII protein supercomplex from Methanosarcina acetivorans . The structure reveals an unprecedented assembly of three NifDK heterotetramers bridged by six NifI _1,2 heterotrimeric PII complexes, which sterically block NifH association and lock the enzyme in an inactive state. The PII complexes display asymmetric binding of ADP and 2-oxoglutarate, coupling nitrogenase inhibition directly to cellular energy and nitrogen status. Addition of 2-oxoglutarate and ATP releases the NifI complexes, stimulating a threefold increase in NifDK activity in vitro . This higher-order architecture uncovers a previously unrecognized regulatory strategy in methanogens, in which PII proteins drive nitrogenase oligomerization to control activity. The discovery that nitrogenase activity may be modulated through direct assembly into higher-order structures opens new avenues for exploring nitrogenase evolution, regulation, and biotechnological applications. One sentence summary: Discovery of a nitrogenase-PII protein supercomplex in methanogens, uncovering a metabolite-gated assembly mechanism for nitrogenase inhibition.

Enzymes that catalyze long-range electron transfer (ET) reactions often function as higher order complexes that possess two structurally symmetrical halves. The functional advantages for such an architecture remain a mystery. Using cryoelectron microscopy we capture snapshots of the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) during substrate binding and turnover. DPOR catalyzes reduction of the C17 = C18 double bond in protochlorophyllide during the dark chlorophyll biosynthetic pathway. DPOR is composed of electron donor (L-protein) and acceptor (NB-protein) component proteins that transiently form a complex in the presence of ATP to facilitate ET. NB-protein is an α₂β₂ heterotetramer with two structurally identical halves. However, our structures reveal that NB-protein becomes functionally asymmetric upon substrate binding. Asymmetry results in allosteric inhibition of L-protein engagement and ET in one half. Residues that form a conduit for ET are aligned in one half while misaligned in the other. An ATP hydrolysis-coupled conformational switch is triggered once ET is accomplished in one half. These structural changes are then relayed to the other half through a di-nuclear copper center at the tetrameric interface of the NB-protein and leads to activation of ET and substrate reduction. These findings provide a mechanistic blueprint for regulation of long-range electron transfer reactions.

Enzymes that catalyze long-range electron transfer reactions are often structurally evolved to possess two symmetrical halves. The functional advantages and mechanistic principles for such architecture remain a mystery. Using Cryo-EM we capture snapshots of the nitrogenase-like Dark-operative Protochlorophyllide Oxidoreductase (DPOR) enzyme during substrate recognition and turnover. The structures reveal that asymmetry is enforced upon substrate binding and leads to an allosteric inhibition of protein-protein interactions and electron transfer in one half. Residues that form a conduit for electron transfer are aligned in one half while misaligned in the other. An ATP-turnover coupled switch is triggered once electron transfer is accomplished in one half and relayed through a bi-copper cluster at the oligomeric interface, leading to activation of enzymatic events in the other. The findings provide a mechanistic blueprint for regulation of asymmetric long-range electron transfer. One-Sentence Summary A bi-copper cluster coordinates electron transfer for substrate reduction in the nitrogenase-like DPOR enzyme and the structures reveal how allostery and asymmetry are enacted over 100Å and utilized for sequential electron transfer.

The 100-year-old Diels–Alder reaction (DAr) is an atom economic and elegant organic chemistry transformation combining a 1,3-diene and a dienophile in a [4+2] cycloaddition leading to a set of products with several stereo centres and multiple stereoisomers. Stereoselective [4+2] cycloaddition is a challenge. Here, we describe two natural enzymes, PyrI4 and AbnU performing stereospecific intermolecular DAr on non-natural substrates. AbnU catalyses a single exo-stereoisomer by 32-fold higher than the background. PyrI4 catalyses the same stereoisomer (15-fold higher) as a major component (>50%). Structural, biochemical and fluorescence studies indicate that the dienophile enters first into the β-barrel of the enzymes followed by the 1,3-diene, yielding a stereospecific product. However, if some critical interactions are disrupted to increase the catalytic efficiency, stereoselectivity is compromised. Since it is established that natural enzymes can carry out intermolecular DAr on non-natural substrates, several hundreds of Diels-Alderases available in nature could be explored.