Electron Transfer Control in Soluble Methane Monooxygenase

The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.National Institutes of Health (U.S.) (Grant GM032134)Waters Corporatio

Conformational Locking upon Cooperative Assembly of Notch Transcription Complexes

SummaryThe Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The “RAM” region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly of a MAML1/NICD/CSL complex using HX-MS. Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2-3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired-site DNA

Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry

This is an Accepted Manuscript of an article published by Taylor & Francis in International Reviews in Physical Chemistry on 2013-1-1, available online: http://www.tandfonline.com/10.1080/0144235X.2012.751175.Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion

Conformational Transitions in the Membrane Scaffold Protein of Phospholipid Bilayer Nanodiscs

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest

Conformational Analysis of Membrane Proteins in Phospholipid Bilayer Nanodiscs by Hydrogen Exchange Mass Spectrometry

The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment. Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a native-like lipid bilayer. Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins. Gamma-glutamyl carboxylase (GGCX), an ~94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions. Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX. Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins

Robust estimation of microbial diversity in theory and in practice

Quantifying diversity is of central importance for the study of structure, function and evolution of microbial communities. The estimation of microbial diversity has received renewed attention with the advent of large-scale metagenomic studies. Here, we consider what the diversity observed in a sample tells us about the diversity of the community being sampled. First, we argue that one cannot reliably estimate the absolute and relative number of microbial species present in a community without making unsupported assumptions about species abundance distributions. The reason for this is that sample data do not contain information about the number of rare species in the tail of species abundance distributions. We illustrate the difficulty in comparing species richness estimates by applying Chao's estimator of species richness to a set of in silico communities: they are ranked incorrectly in the presence of large numbers of rare species. Next, we extend our analysis to a general family of diversity metrics ("Hill diversities"), and construct lower and upper estimates of diversity values consistent with the sample data. The theory generalizes Chao's estimator, which we retrieve as the lower estimate of species richness. We show that Shannon and Simpson diversity can be robustly estimated for the in silico communities. We analyze nine metagenomic data sets from a wide range of environments, and show that our findings are relevant for empirically-sampled communities. Hence, we recommend the use of Shannon and Simpson diversity rather than species richness in efforts to quantify and compare microbial diversity.Comment: To be published in The ISME Journal. Main text: 16 pages, 5 figures. Supplement: 16 pages, 4 figure

Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multisubstrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structureguided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of similar to 500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A