Hui Qi Ng

NMR structural characterization of the N-terminal active domain of the gyrase B subunit from Pseudomonas aeruginosa and its complex with an inhibitor.

The N‐terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) fromPseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and abis‐pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targetingP. aeruginosa.

Insight into the molecular interaction between the cyclic nucleotide-binding homology domain and the eag domain of the hERG channel

The gating of the hERG channel is regulated by its eag domain through molecular interaction with either the cyclic nucleotide‐binding homology domain (CNBHD) or the linker between transmembrane segments 4 and 5. Our NMR study on the purified CNBHD demonstrated that it contains nine β‐strands and does not bind cAMP. We show that the eag domain binds to the CBND through an interface containing several disease‐associated mutations. The N‐terminal cap domain and R56 in the eag domain are important for the interaction with the CNBHD. Residues from the CNBHD that were affected by the interaction with the eag domain were also identified. A R56Q mutation does not cause major structural changes in the eag domain and showed reduced interaction with the CNBHD.

Purification and structural characterization of the voltage-sensor domain of the hERG potassium channel

The hERG (human ether à go-go related gene) potassium channel is a voltage-gated potassium channel playing important roles in the heart by controlling the rapid delayed rectifier potassium current. The hERG protein contains a voltage-sensor domain (VSD) that is important for sensing voltage changes across the membrane. Mutations in this domain contribute to serious heart diseases. To study the structure of the VSD, it was over-expressed in Escherichia coli and purified into detergent micelles. Lyso-myristoyl phosphatidylglycerol (LMPG) was shown to be a suitable detergent for VSD purification and folding. Secondary structural analysis using circular dichroism (CD) spectroscopy indicated that the purified VSD in LMPG micelles adopted α-helical structures. Purified VSD in LMPG micelles produced dispersed cross-peaks in a (15)N-HSQC spectrum. Backbone resonance assignments for residues from transmembrane segments S3 and S4 of VSD also confirmed the presence of α-helical structures in this domain. Our results demonstrated that structure of VSD can be investigated using NMR spectroscopy.

Solution structure of the cyclic-nucleotide binding homology domain of a KCNH channel

The carboxy-terminal region of the KCNH family of potassium channels contains a cyclic-nucleotide binding homology domain (CNBHD) that is important for channel gating and trafficking. The solution structure of the CNBHD of the KCNH potassium of zebrafish was determined using solution NMR spectroscopy. This domain exists as a monomer under solution conditions and adopts a similar fold to that determined by X-ray crystallography. The CNBHD does not bind cAMP because residue Y740 blocks the entry of cyclic-nucleotide to the binding pocket. Relaxation results show that the CNBHD is rigid except that some residues in the loop between β6 and β7 are flexible. Our results will be useful to understand the gating mechanism of KCNH family members through the CNBHD.

Escherichia coli topoisomerase IV E subunit and an inhibitor binding mode revealed by NMR spectroscopy

Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of β1-α2 and residues in the loop of β3-β4 might improve the inhibitor potency.

Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel

The hERG (human ether‐a‐go‐go related gene) potassium channel is a voltage‐gated potassium channel containing an N‐terminal domain, a voltage‐sensor domain, a pore domain and a C‐terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2–S3 linker, S3, S4 and the S4–S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso‐myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4–S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3–S4 and S4–S4–S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating. Copyright

1H, 13C and 15N chemical shift assignments for the N-terminal PAS domain of the KCNH channel from Zebrafish

The KCNH channels are voltage-gated potassium channels that play important roles in heart and nerve cells. The N-terminal region of the KCNH channel contains a Per-Arnt-Sim (PAS) domain which is important for the channel gating through interaction with other regions of the channel. To study the solution structure of the N-terminal PAS domain of the KCNH channel from Zebrafish (zNTD), we over-expressed and purified zNTD. We report the resonance assignments for zNTD. The data will allow us to perform structural studies for this domain, which will provide insight into its structural basis for the molecular interaction with other regions of the KCNH channel.

Structure of the Cyclic Nucleotide-Binding Homology Domain of the hERG Channel and Its Insight into Type 2 Long QT Syndrome

The human ether-à-go-go related gene (hERG) channel is crucial for the cardiac action potential by contributing to the fast delayed-rectifier potassium current. Mutations in the hERG channel result in type 2 long QT syndrome (LQT2). The hERG channel contains a cyclic nucleotide-binding homology domain (CNBHD) and this domain is required for the channel gating though molecular interactions with the eag domain. Here we present solution structure of the CNBHD of the hERG channel. The structural study reveals that the CNBHD adopts a similar fold to other KCNH channels. It is self-liganded and it contains a short β-strand that blocks the nucleotide-binding pocket in the β-roll. Folding of LQT2-related mutations in this domain was shown to be affected by point mutation. Mutations in this domain can cause protein aggregation in E. coli cells or induce conformational changes. One mutant-R752W showed obvious chemical shift perturbation compared with the wild-type, but it still binds to the eag domain. The helix region from the N-terminal cap domain of the hERG channel showed unspecific interactions with the CNBHD.

1 H, 13 C and 15 N chemical shift assignments for the cyclic-nucleotide binding homology domain of a KCNH channel

The KCNH family of ion channels plays important roles in heart and nerve cells. The C-terminal region of the KCNH channel contains a cyclic-nucleotide binding homology domain (CNBHD) which is important for channel gating through interaction with the eag domain. To study the solution structure of CNBHD of the KCNH channel of zebrafish, we over-expressed and purified this domain from E. coli. We report the resonance assignments of the CNBHD. The assignments will allow us to perform structural and dynamic studies for this domain, which will shed light on its role in channel gating.

Backbone resonance assignments for the SET domain of human methyltransferase NSD3 in complex with its cofactor

NSD3 is a histone H3 methyltransferase that plays an important role in chromatin biology. A construct containing the methyltransferase domain encompassing residues Q1049-K1299 of human NSD3 was obtained and biochemical activity was demonstrated using histone as a substrate. Here we report the backbone HN, N, Cα, C′, and side chain Cβ assignments of the construct in complex with S-adenosyl-l-methionine (SAM). Based on these assignments, secondary structures of NSD3/SAM complex in solution were determined.