Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State

NMR structures of membrane proteins are often hampered by poor chemical shift dispersion and internal dynamics which limit resolved distance restraints. However, the ordering and topology of these systems can be defined with site-specific water or lipid proximity. Membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here we leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine a membrane protein structure. This is demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. We measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1–300 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.

Analysis of changes of cavity volumes in predefined directions of protein motions and cavity flexibility

Dynamics of protein cavities associated with protein fluctuations and conformational plasticity is essential for their biological function. NMR ensembles, Molecular Dynamics (MD) simulations combined with Principal Component Analysis (PCA), and Normal Mode Analysis (NMA) provide appropriate frameworks to explore functionally relevant protein dynamics and cavity changes relationships. Within this context, we have developed ANA (Analysis of Null Areas), an efficient method to calculate cavity volumes. ANA is based on a combination of algorithms that guarantees its robustness against numerical differentiations. This is a unique feature with respect to other methods. Herein, we test ANA as a biophysical and bioinformatic method to analyze different structural and dynamics properties of cavities. In order to address this task, we have developed an updated and improved version of ANA that expands it use to quantify changes in cavity features, like volume and flexibility, due to protein structural distortions performed on predefined biologically relevant directions, e.g, directions of largest contribution to protein fluctuations (PCA modes) obtained by MD simulations or ensembles of NMR structures, collective NMA modes or any other direction of motion associated with specific conformational changes. A web page has been developed and its facilities are explained in detail, making the software available to all users. Firstly, we show that ANA can be useful to explore gradual changes of cavity volume and flexibility associated with protein ligand binding. Secondly, we perform a comparison study of the extent of variability between protein backbone structural distortions, and changes in cavity volumes and flexibilities evaluated for an ensemble of NMR active and inactive conformers of the epidermal growth factor receptor (EGFR) structures. Finally, we compare changes in size and flexibility between sets of NMR structures for different homologous chains of dynein.

Resonance assignment and secondary structure of DbpA protein from the European species, Borrelia afzelii

Decorin binding proteins (Dbps) mediate attachment of spirochetes in host organisms during the early stages of Lyme disease infection. Previously, different binding mechanisms of Dbps to glycosaminoglycans have been elucidated for the pathogenic species Borrelia burgdorferi sensu stricto and B. afzelii. We are investigating various European Borrelia spirochetes and their interactions at the atomic level using NMR. We report preparative scale recombinant expression of uniformly stable isotope enriched B. afzelii DbpA in Escherichia coli, its chromatographic purification, and solution NMR assignments of its backbone and sidechain 1H, 13C, and 15N atoms. This data was used to predict secondary structure propensity, which we compared to the North American B. burgdorferi sensu stricto and European B. garinii DbpA for which solution NMR structures had been determined previously. Backbone dynamics of DbpA from B. afzelii were elucidated from spin relaxation and heteronuclear NOE experiments. NMR-based secondary structure analysis together with the backbone dynamics characterization provided a first look into structural differences of B. afzelii DbpA compared to the North American species and will serve as the basis for further investigation of how these changes affect interactions with host components.

Structures of the ApoL1 and ApoL2 N-terminal domains reveal a non-classical four-helix bundle motif

Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.

SenseNet, a tool for analysis of protein structure networks obtained from molecular dynamics simulations

Computational methods play a key role for investigating allosteric mechanisms in proteins, with the potential of generating valuable insights for innovative drug design. Here we present the SenseNet ("Structure ENSEmble NETworks") framework for analysis of protein structure networks, which differs from established network models by focusing on interaction timelines obtained by molecular dynamics simulations. This approach is evaluated by predicting allosteric residues reported by NMR experiments in the PDZ2 domain of hPTP1e, a reference system for which previous computational predictions have shown considerable variance. We applied two models based on the mutual information between interaction timelines to estimate the conformational influence of each residue on its local environment. In terms of accuracy our prediction model is comparable to the top performing model published for this system, but by contrast benefits from its independence from NMR structures. Our results are complementary to experimental data and the consensus of previous predictions, demonstrating the potential of our new analysis tool SenseNet. Biochemical interpretation of our model suggests that allosteric residues in the PDZ2 domain form two distinct clusters of contiguous sidechain surfaces. SenseNet is provided as a plugin for the network analysis software Cytoscape, allowing for ease of future application and contributing to a system of compatible tools bridging the fields of system and structural biology.

Role of backbone strain in de novo design of complex α/β protein structures

We previously elucidated principles for designing ideal proteins with completely consistent local and non-local interactions which have enabled the design of a wide range of new αβ-proteins with four or fewer β-strands. The principles relate local backbone structures to supersecondary-structure packing arrangements of α-helices and β-strands. Here, we test the generality of the principles by employing them to design larger proteins with five- and six- stranded β-sheets flanked by α-helices. The initial designs were monomeric in solution with high thermal stability, and the nuclear magnetic resonance (NMR) structure of one was close to the design model, but for two others the order of strands in the β-sheet was swapped. Investigation into the origins of this strand swapping suggested that the global structures of the design models were more strained than the NMR structures. We incorporated explicit consideration of global backbone strain into the design methodology, and succeeded in designing proteins with the intended unswapped strand arrangements. These results illustrate the value of experimental structure determination in guiding improvement of de novo design, and the importance of consistency between local, supersecondary, and global tertiary interactions in determining protein topology. The augmented set of principles should inform the design of larger functional proteins.

Biophysical characterization of the SARS-CoV-2 E protein

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic which continues to wreak havoc across the world, over a year and a half after its effects were first reported in the general media. Current fundamental research efforts largely focus on the SARS-CoV-2 Spike protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length E protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

Structure and antimicrobial activity of NCR169, a nodule-specific cysteine-rich peptide of Medicago truncatula

A model legume, Medicago truncatula, has over 600 nodule-specific cysteine-rich (NCR) peptides required for symbiosis with rhizobia. Among them, NCR169, an essential factor for establishing symbiosis, has four cysteine residues that are indispensable for its function. However, knowledge of NCR169 structure and mechanism of action is still lacking. In this study, we solved two NMR structures of NCR169 caused by different disulfide linkage patterns. We show that both structures have a consensus C-terminal β-sheet attached to an extended N-terminal region with dissimilar features; one moves widely, whereas the other is relatively stapled. We further revealed that the disulfide bonds of NCR169 contribute to its structural stability and solubility. Regarding the function, one of the NCR169 oxidized forms could bind to negatively charged bacterial phospholipids. Furthermore, the positively charged lysine-rich region of NCR169 may be responsible for its antimicrobial activity against Escherichia coli and Sinorhizobium meliloti. This active region was disordered even in the phospholipid bound state, suggesting that the disordered conformation of this region is key to its function. Morphological observations suggested the mechanism of action of NCR169 on bacteria. The present study on NCR169 provides new insights into the structure and function of NCR peptides.

The accuracy of NMR protein structures in the Protein Data Bank

We recently described a method, ANSURR, for measuring the accuracy of NMR protein structures. It is based on comparing residue-specific measures of rigidity from backbone chemical shifts via the random coil index, and from structures. Here, we report the use of ANSURR to analyse NMR ensembles within the Protein Data Bank (PDB). NMR structures cover a wide range of accuracy, which improved over time until about 2005, since when accuracy has not improved. Most structures have accurate secondary structure, but are too floppy, particularly in loops. There is a need for more experimental restraints in loops. The best current accuracy measures are Ramachandran distribution and number of NOE restraints per residue. The precision of structure ensembles correlates with accuracy, as does the number of hydrogen bond restraints per residue. If a structure contains additional components (such as additional polypeptide chains or ligands), then their inclusion improves accuracy. Analysis of over 7000 PDB NMR ensembles is available via our website ansurr.com.