Significance GabR is a member of the MocR/GabR subfamily of the GntR family of bacterial transcription regulators. It regulates the metabolism of γ-aminobutyric acid, an important nitrogen and carbon source in many bacteria. The crystal structures reported here show that this protein has evolved from the fusion of a type I aminotransferase and a winged helix-turn-helix DNA-binding protein to form a chimeric protein that adopts a dimeric head-to-tail configuration. The pyridoxal 5′-phosphate–binding regulatory domain of GabR is therefore an example of a coenzyme playing a role in transcription regulation rather than in enzymatic catalysis. Our structural and biochemical studies lay the mechanistic foundation for understanding the regulatory functions of the MocR/GabR subfamily of transcription regulators.

Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (Roc R1441H ) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.M utation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD) (1-5). LRRK2 is a large (2,527-aa) multidomain protein consisting of seven putative domains (2), including a Ras-like GTPase domain called Ras of complex proteins (Roc), followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear how perturbations of these activities result in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild type; therefore, its overactivation might be associated with disease pathogenesis (6).The tandem Roc-COR-Kin arrangement suggests that their activities might be coupled such that the GTPase activity of Roc might modulate the kinase activity. Indeed, several studies have shown that GTP binding to the Roc domain regulates the activity of the Kin domain (7,8). Moreover, a PD-associated mutation in the Roc domain (R1441C) has been shown to have higher kinase activity (9), thus suggesting that mutations in the Roc domain, also up-regulate kinase activity.Understanding the function of Roc and its mechanism of action is important for understanding the mechanism of PD pathogenesis and therapeutic development. However, because of the lack sufficient quantity of protein samples amendable for detailed investigations, the biochemical properties and enzymatic activities of the Roc domain of LRRK2 are poorly understood.Here, we describe a stably folded construct of human Roc domain that enabled us to investigate quantitatively its biochemical and enzymatic properties. The results revealed that a PD-causing mutation R1441H in the Roc domain renders it less active at hydrolyzing GTP, as well as having higher affinity for GTP, than its wild-type counterpart, thereby increasing the residence time of its GTP-bound "active state," which is associated with PD pathogenesis (8). Results and DiscussionConstruction of a Stable Human LRR...

SummaryChromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein’s structure rather than that of the DNA template.

Abstract. Room temperature (300 K) experiments have earlier established that the replacement of a fluorine atom in p-difluorobenzene (pDFB) with a methyl group to make p-fluorotoluene @FT) generates a qualitative difference in intramolecular vibrational redistribution (IVR) characteristics as seen in the S, states. Here we report S , S , fluorescence excitation and dispersed single vibronic level (SVL) fluorescence spectra that have been obtained for IVR comparisons in the cold (5 K) environment of supersonic expansions. As in the 300 K experiments, the cold beam S, vibrational energy threshold for IVR is substantially lower in pFT. The vibrational congestion in dispersed fluorescence that reveals extensive S, level interactions first appears after pumping an S, pFT level near 800 cm-'. In contrast, congestion in pDFB spectra is still absent from levels with twice this energy. Attention is directed to the relative S, ring level structures as a potential contributor to the distinctive IVR behaviors. Dispersed fluorescence from the S, zero point levels and fluorescence excitation spectra are combined with published information about S, fundamentals to show that the S, vibrational level structures of the two molecules are as closely related as those of an isotopomer pair. It is argued that the small differences in S, fundamentals cannot be a principal contribution to the qualitative IVR differences.

To gain insight into the possible magnitude of vibrational activation/deactivation rate constants for large molecules with the high vibrational excitation of thermal unimolecular reactions, absolute vibrational relaxation rate constants have been measured in S1 p-difluorobenzene energy regions where the vibrational levels begin to form a quasi-continuum. The measured rate constants define vibrational energy transfer from three initially pumped S1 levels into the surrounding vibrational field. The observed rate constants are about 60% of the Lennard-Jones value for the collision partner Ar and about 40% for He. The initial levels lie in the εvib range 2887 to 3310 cm-1, where the level densities are approximately 800−2000 per cm-1. New He rate constants are also measured for many lower initial levels. When combined with earlier measurements, the He and Ar rate constants span the range 0 ≤ εvib ≤ 3310 cm-1. Both sets of rate constants show a trend to larger values with increasing εvib and have prominent variations in response to the zero order quantum identity of the initial level. When the high level density starts to create overlapping states, the Ar rate constants for the three highest levels appear to have leveled off, and the initial quantum state variations are damped out. The He rate constants, on the other hand, sustain the trend to larger values even at the highest energies where the data are ambiguous on whether initial quantum state sensitivity persists.

Summary Clathrin is a trimeric protein involved in receptor-mediated-endocytosis, but can function as a non-trimer outside of endocytosis. We have discovered that the subcellular distribution of a clathrin cysteine mutant we previously studied is altered and a proportion is also localized to nuclear spaces. MALS shows C1573A hub is a mixture of trimer-like and detrimerized molecules. The X-ray structure of the trimerization domain reveals that that without light chains, a helix harboring cysteine-1573 is reoriented. We propose clathrin has a detrimerization switch, which suggests clathrin topology can be altered naturally for new functions.

No abstract

Glycosylation is a common post-translational modification and critical quality attribute that can modulate the efficacy of therapeutic proteins. In the production of monoclonal antibodies (mAbs), quantifying the glycoform profile is a vital characterization step. Traditional glycan analysis is time consuming and involves steps at extreme temperature or pH, which may alter glycans. Here, we describe a rapid method for glycan analysis in which glycans are released from mAb samples that are bound to protein A columns. Since host cell proteins, which may also contain glycans, were already removed, this step enables analysis of cell culture products. Glycans released from the mAb samples are then derivatized with InstantPC™ labeling agent and analyzed by HILIC-FLD-MS. To illustrate the method, the glycan profiles of six trastuzumab (Herceptin®) antibody lots and four biosimilar developmental lots were analyzed. The results derived from our novel method, which takes less than 90 min, are compared with those from a typical glycan preparation approach.