NMR solution structure of a chymotrypsin inhibitor from the Taiwan cobra Naja naja atra

The Taiwan cobra (Naja naja atra) chymotrypsin inhibitor (NACI) consists of 57 amino acids and is related to other Kunitz-type inhibitors such as bovine pancreatic trypsin inhibitor (BPTI) and Bungarus fasciatus fraction IX (BF9), another chymotrypsin inhibitor. Here we present the solution structure of NACI. We determined the NMR structure of NACI with a root-mean-square deviation of 0.37 Å for the backbone atoms and 0.73 Å for the heavy atoms on the basis of 1,075 upper distance limits derived from NOE peaks measured in its NOESY spectra. To investigate the structural characteristics of NACI, we compared the three-dimensional structure of NACI with BPTI and BF9. The structure of the NACI protein comprises one 310-helix, one α-helix and one double-stranded antiparallel β-sheet, which is comparable with the secondary structures in BPTI and BF9. The RMSD value between the mean structures is 1.09 Å between NACI and BPTI and 1.27 Å between NACI and BF9. In addition to similar secondary and tertiary structure, NACI might possess similar types of protein conformational fluctuations as reported in BPTI, such as Cys14–Cys38 disulfide bond isomerization, based on line broadening of resonances from residues which are mainly confined to a region around the Cys14–Cys38 disulfide bond

Regulation of the Activity in the p53 Family Depends on the Organization of the Transactivation Domain.

Despite high sequence homology among the p53 family members, the regulation of their transactivation potential is based on strikingly different mechanisms. Previous studies revealed that the activity of TAp63α is regulated via an autoinhibitory mechanism that keeps inactive TAp63α in a dimeric conformation. While all p73 isoforms are constitutive tetramers, their basal activity is much lower compared with tetrameric TAp63. We show that the dimeric state of TAp63α not only reduces DNA binding affinity, but also suppresses interaction with the acetyltransferase p300. Exchange of the transactivation domains is sufficient to transfer the regulatory characteristics between p63 and p73. Structure determination of the transactivation domains of p63 and p73 in complex with the p300 Taz2 domain further revealed that, in contrast to p53 and p73, p63 has a single transactivation domain. Sequences essential for stabilizing the closed dimer of TAp63α have evolved into a second transactivation domain in p73 and p53.The research was funded by the DFG (DO 545/8 and DO 545/13), the Center for Biomolecular Magnetic Resonance (BMRZ), and the Cluster of Excellence Frankfurt (Macromolecular Complexes). M.T. was supported by a fellowship from the Fonds of the Chemical Industry

Rapid protein assignments and structures from raw NMR spectra with the deep learning technique ARTINA

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the major techniques in structural biology with over 11,800 protein structures deposited in the Protein Data Bank. NMR can elucidate structures and dynamics of small and medium size proteins in solution, living cells, and solids, but has been limited by the tedious data analysis process. It typically requires weeks or months of manual work of a trained expert to turn NMR measurements into a protein structure. Automation of this process is an open problem, formulated in the field over 30 years ago. Here, we present a solution to this challenge that enables the completely automated analysis of protein NMR data within hours after completing the measurements. Using only NMR spectra and the protein sequence as input, our machine learning-based method, ARTINA, delivers signal positions, resonance assignments, and structures strictly without any human intervention. Tested on a 100-protein benchmark comprising 1329 multidimensional NMR spectra, ARTINA demonstrated its ability to solve structures with 1.44 {\AA} median RMSD to the PDB reference and to identify 91.36% correct NMR resonance assignments. ARTINA can be used by non-experts, reducing the effort for a protein assignment or structure determination by NMR essentially to the preparation of the sample and the spectra measurements

Automated protein structure calculation from NMR data

Current software is almost at the stage to permit completely automatic structure determination of small proteins of < 15 kDa, from NMR spectra to structure validation with minimal user interaction. This goal is welcome, as it makes structure calculation more objective and therefore more easily validated, without any loss in the quality of the structures generated. Moreover, it releases expert spectroscopists to carry out research that cannot be automated. It should not take much further effort to extend automation to ca 20 kDa. However, there are technological barriers to further automation, of which the biggest are identified as: routines for peak picking; adoption and sharing of a common framework for structure calculation, including the assembly of an automated and trusted package for structure validation; and sample preparation, particularly for larger proteins. These barriers should be the main target for development of methodology for protein structure determination, particularly by structural genomics consortia

Robust Symbol Level Precoding Designs in Multiuser MIMO Systems

Multiple-user (MU) multiple-input multiple-output (MIMO) technology, which involves using multiple antennas to simultaneously serve multiple users or devices, is a cornerstone of both 5G and 6G. The use of MIMO in ultra-dense networks with smaller cell sizes and more antennas will result in a proportional increase in both inter- and intra-cell interference. To manage the interference, precoding or beamforming is needed to steer the transmit signals towards intended users and mitigate interference. Symbol level precoding (SLP) techniques exploit information about the symbols to be transmitted in addition to the channel state information (CSI), which can significantly improve performance at the expense of increased complexity at the transmitter. The additional degrees of freedom (DoF) provided by the symbol-level information make it possible to exploit the constructive component of the interference, converting it into constructive interference (CI) that can move the received signals further from the decision thresholds of the constellation points. CI-based SLP recasts the traditional viewpoint of interference as a source of degradation to one where interference is a potential resource that can be exploited. In this dissertation, we firstly study the use of SLP in the downlink of a multiuser multiple-input-single-output (MU-MISO) cognitive radio (CR) network, where a primary base station (PBS) serving primary users (PUs) and a cognitive base station (CBS) serving cognitive users (CUs) share the same frequency band. The SLP approach is designed using the symbol-wise Maximum Safety Margin (MSM) criterion, which exploits the constructive multiuser interference present in such a network. We adapt the non-linear MSM precoder to both underlay and overlay CR scenarios, depending on whether or not the primary system shares its information with the cognitive system. Secondly, we investigate robust SLP designs in an overlay CR network, where the primary and secondary networks transmit signals concurrently, however, the PBS shares imperfect CSI with the CBS. We propose robust SLP schemes in this scenario and consider two different CSI error models. For the norm-bounded CSI error model, we adopt a max-min philosophy to conservatively achieve robust SLP constraints; for the additive quantization noise model (AQNM), we employ a stochastic constraint to formulate the problem. Simulation results show that, rather than simply trying to eliminate the network's cross-interference, the proposed robust SLP schemes enable the primary and secondary networks to aid each other in meeting their quality of service constraints. Moreover, we propose precoding design in multi-antenna systems with improper Gaussian interference (IGI), characterized by correlated real and imaginary parts. We first study block level precoding (BLP) and SLP assuming the receivers apply a pre-whitening filter to decorrelate and normalize the IGI. We then shift to the scenario where the base station (BS) incorporates the IGI statistics in the SLP design, which allows the receivers to employ a standard detection algorithm without pre-whitenting. Finally we address the case where the non-circularity of the IGI is unknown, and we formulate robust BLP and SLP designs that minimize the worst case performance in such settings. Interestingly, we show that for BLP, the worst-case IGI is in fact proper, while for SLP the worst case occurs when the interference signal is maximally improper, with fully correlated real and imaginary parts. The numerical results reveal the superior performance of SLP in terms of symbol error rate (SER) and energy efficiency (EE), especially for the case where there is uncertainty in the non-circularity of the jammer

NMR structure of the protein NP_247299.1: comparison with the crystal structure

Comparison of the NMR and crystal structures of a protein determined using largely automated methods has enabled the interpretation of local differences in the highly similar structures. These differences are found in segments of higher B values in the crystal and correlate with dynamic processes on the NMR chemical shift timescale observed in solution

NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations

Cold-Adapted Signal Proteins: NMR Structures of Pheromones from the Antarctic Ciliate Euplotes nobilii

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear 1H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-a-helix bundle with an up-down-up topology and a 310-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones.TheNMRstructures ofEn-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters

Structural and functional characterization of MuB protein involved in DNA targeting for transposition

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 02-09-2014DNA transposons are ubiquitous in the genomes of all forms of life and play important evolutionary roles in generating gene diversity and in shaping genomic landscapes. Mu phage is one of the most complex and efficient DNA transposon. Mu transposition requires two phage-­‐encoded proteins: the transposase MuA and the accessory protein MuB. MuB is an ATP-­‐dependent non-­‐specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by its poor solubility and tendency to aggregate. We combined bioinformatic, mutagenic, biochemical, electron microscopic and NMR analyses to unmask the structure and function of MuB. We demonstrate that MuB is an AAA+ ATPase composed of an N-­‐terminal appendage and an AAA+ ATPase module that upon ATP binding forms helical filaments on the DNA. We also identify critical residues for its ATPase, DNA binding, protein polymerization and MuA interaction activities. Using single-­‐particle electron microscopy, we show that MuB assembles into helical filaments that coat the DNA without deforming it, resulting in a unique protein-­‐DNA symmetry mismatch. These findings, together with the influence of MuB-­‐filament size on strand-­‐transfer efficiency, lead to a model in which MuB-­‐ imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition. We have also observed the tendency of the MuB filaments to form bundles in an N-­‐terminal appendage dependent manner. The structure of the N-­‐terminal appendage was solved by NMR spectroscopy. The N-­‐terminal appendage is strikingly similar to the λ-­‐ repressor like DNA-­‐binding domains, strongly suggesting that this MuB domain could be involved in DNA recognition. This led us to propose a new model of Mu phage target immunity in which filament-­‐filament interactions mediated by the N-­‐terminal appendage could aid in the condensation of the phage DNA, occluding it from the transposase.Los transposones de ADN son ubicuos en los genomas de todos los seres vivos y juegan un papel evolutivo importante en la generación de diversidad génica y en la definición de los genomas. El bacteriófago Mu es uno de los transposones de ADN más complejos y efectivos. La transposición de Mu requiere dos proteínas codificadas por el fago: la transposasa MuA y la proteína accesoria MuB. MuB es una proteína de unión a ADN dependiente de ATP que regula la actividad de la transposasa y captura el ADN diana para la transposición. La comprensión de la función de MuB a nivel mecanístico se ha visto dificultada por su baja solubilidad y su tendencia a agregar. Hemos combinado análisis bioinformatico, mutagénesis, bioquímica, microscopía electrónica y NMR para desenmascarar la estructura y la función de MuB. Demostramos que MuB es una ATPasa AAA+ compuesta de un apéndice N-­‐terminal y un módulo AAA+ que al unir ATP forma filamentos helicoidales sobre el ADN. También hemos identificado residuos clave para la unión e hidrólisis del ATP, unión al ADN, polimerización e interacción con MuA. Usando miscroscopía electrónica de partículas individuales mostramos que MuB se ensambla en filamentos helicoidales que cubren el ADN sin deformarlo, resultando en un mal emparejamiento único. Estos resultados, junto a resultados de cómo el tamaño de los filamentos de MuB afecta a la eficiencia de la transposición, sugieren un modelo según el cual la simetría impuesta por el filamento de MuB deforma transitoriamente el ADN al final del filamento, presentándolo como un mejor sustrato para la transposasa MuA. Hemos observado también la tendencia de los filamentos de MuB a formar haces de un modo que depende del apéndice N-­‐ terminal. Hemos resuelto la estructura del apéndice N-­‐terminal mediante espectroscopía de RMN. El apéndice N-­‐terminal es sorprendentemente similar a los dominios de unión de ADN de la familia del represor λ, sugiriendo que este dominio de MuB podría estar implicado en el reconocimiento del ADN. Estos resultados nos llevan a proponer un nuevo mecanismo de inmunidad del fago Mu, en el que las interacciones entre filamentos mediadas por el apéndice N-­‐terminal podrían ayudar a la condensación del genoma de Mu, ocultándolo así de la acción de la transposasa. xvii

Protein and metal cluster structure of the wheat metallothionein domain γ-Ec-1: the second part of the puzzle

Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including ZnII, CdII, and CuI. MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT Ec-1 from Triticum aestivum, common bread wheat, is a ZnII-binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding βEdomain was recently described. Here we present the structure of the N-terminal second domain, γ-Ec-1, determined by NMR spectroscopy. The γ-Ec-1 domain enfolds an M 2 II Cys6 cluster and was characterized as part of the full-length Zn6Ec-1 protein as well as in the form of the separately expressed domain, both in the ZnII-containing isoform and the CdII-containing isoform. Extended X-ray absorption fine structure analysis of Zn2γ-Ec-1 clearly shows the presence of a ZnS4 coordination sphere with average Zn-S distances of 2.33Å. 113CdNMR experiments were used to identify the MII-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of γ-Ec-1 were probed with CdII binding experiments as well as by pH titrations of the ZnII and CdII forms, the latter suggesting an interaction of the γdomain and the βEdomain within the full-length protei