Nitrogenase beyond the Resting State: A Structural Perspective.

Nitrogenases have the remarkable ability to catalyze the reduction of dinitrogen to ammonia under physiological conditions. How does this happen? The current view of the nitrogenase mechanism focuses on the role of hydrides, the binding of dinitrogen in a reductive elimination process coupled to loss of dihydrogen, and the binding of substrates to a binuclear site on the active site cofactor. This review focuses on recent experimental characterizations of turnover relevant forms of the enzyme determined by cryo-electron microscopy and other approaches, and comparison of these forms to the resting state enzyme and the broader family of iron sulfur clusters. Emerging themes include the following: (i) The obligatory coupling of protein and electron transfers does not occur in synthetic and small-molecule iron-sulfur clusters. The coupling of these processes in nitrogenase suggests that they may involve unique features of the cofactor, such as hydride formation on the trigonal prismatic arrangement of irons, protonation of belt sulfurs, and/or protonation of the interstitial carbon. (ii) Both the active site cofactor and protein are dynamic under turnover conditions; the changes are such that more highly reduced forms may differ in key ways from the resting-state structure. Homocitrate appears to play a key role in coupling cofactor and protein dynamics. (iii) Structural asymmetries are observed in nitrogenase under turnover-relevant conditions by cryo-electron microscopy, although the mechanistic relevance of these states (such as half-of-sites reactivity) remains to be established.

Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation.

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase.

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and ß-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and ß-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures.

In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.

Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins.

Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.

Structural consequences of turnover-induced homocitrate loss in nitrogenase.

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein - a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA - that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site. We further find a population of the ∆NifV variant complexed to an endogenous protein identified through structural and proteomic approaches as the uncharacterized protein NafT. Recognition by endogenous NafT demonstrates the physiological relevance of the HCA-compromised form, perhaps for cofactor insertion or repair. Our results point towards a dynamic active site in which HCA plays a role in enabling nitrogenase catalysis by facilitating activation of the FeMo-cofactor from a relatively stable form to a state capable of reducing dinitrogen under ambient conditions.

l-Isoaspartyl Methyltransferase Deficiency in Zebrafish Leads to Impaired Calcium Signaling in the Brain.

Isomerization of l-aspartyl and l-asparaginyl residues to l-isoaspartyl residues is one type of protein damage that can occur under physiological conditions and leads to conformational changes, loss of function, and enhanced protein degradation. Protein l-isoaspartyl methyltransferase (PCMT) is a repair enzyme whose action initiates the reconversion of abnormal l-isoaspartyl residues to normal l-aspartyl residues in proteins. Many lines of evidence support a crucial role for PCMT in the brain, but the mechanisms involved remain poorly understood. Here, we investigated PCMT activity and function in zebrafish, a vertebrate model that is particularly well-suited to analyze brain function using a variety of techniques. We characterized the expression products of the zebrafish PCMT homologous genes pcmt and pcmtl. Both zebrafish proteins showed a robust l-isoaspartyl methyltransferase activity and highest mRNA transcript levels were found in brain and testes. Zebrafish morphant larvae with a knockdown in both the pcmt and pcmtl genes showed pronounced morphological abnormalities, decreased survival, and increased isoaspartyl levels. Interestingly, we identified a profound perturbation of brain calcium homeostasis in these morphants. An abnormal calcium response upon ATP stimulation was also observed in mouse hippocampal HT22 cells knocked out for Pcmt1. This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time in vivo, that PCMT plays a pivotal role in the regulation of calcium fluxes.

Structure of amyloid-ß (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface.

Amyloid-ß (Aß) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aß 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt ß-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aß structures, and both self-associate through two distinct interfaces. One of these is a unique Aß interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aß 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aß segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.

Racemized and Isomerized Proteins in Aging Rat Teeth and Eye Lens.

The quantification of aspartic acid racemization in the proteins of nonmetabolically active tissues can be used as a measure of chronological aging in humans and other long-lived organisms. However, very few studies have been conducted in shorter-lived animals such as rodents, which are increasingly used as genetic and metabolic models of aging. An initial study had reported significant changes in the ratio of d- to l-aspartate in rat molars with age. Using a sensitive HPLC method for the determination of d- and l-aspartate from protein hydrolysates, we found no accumulation of d-aspartate in the molars of 17 rats that ranged in age from 2 to 44 months, and the amount of d-aspartate per molar did not correspond with molar eruption date as had been previously reported. However, developing an alternate approach, we found significant accumulation of isomerized aspartyl residues in eye lens proteins that are also formed by spontaneous degradation processes. In this study, we used the human protein l-isoaspartate/d-aspartate O-methyltransferase (PCMT1) as an analytical reagent in a sensitive and convenient procedure that could be used to rapidly examine multiple samples simultaneously. We found levels of isomerized aspartyl residues to be about 35 times higher in the lens extracts of 18-month-old rats versus 2-month-old rats, suggesting that isomerization may be an effective marker for biological aging in this range of ages. Importantly, we found that the accumulation appeared to plateau in rats of 18 months and older, indicating that potentially novel mechanisms for removing altered proteins may develop with age.