RESEARCH PLAN Brian Michael Lee

My research efforts are focused on the three funded projects described below. Other research collaborations include screening antigenic surface-displayed proteins from non-typeable Haemophilus influenza, developing a swine model of rheumatic fever, computational modeling of long-range mRNA interactions based on SHAPE experimental data for the splicing of survival motor neuron (SMN) gene, comparative modeling of SMN protein variants that influence spliceosome assembly, and NMR solution studies of the PDZ domain from the cytoskeletal adaptor protein, LMO7. Project 1: Metal-dependent phosphohydrolase activity and RNA binding specificity of the conserved virulence factor A from Streptococcus pyogenes during post-transcriptional regulation of pathogenesis. Collaborators: Gabriela Pérez-Alvarado (Coastal Carolina University) and Kyu Hong Cho (Indiana State University) Current Funding: NIH-NIGMS (R15GM101603) August 2012 to March 2017 Future Funding: renewal of the NIH-NIGMS R15 with submission planned in June 2017

Specific Aims: Define the role of CvfA in regulating the expression of virulence genes. Aim 1: Structure and RNA binding specificity of the KH domain of CvfA (Lee) Aim 2: Identification of the RNA substrates and the regulatory role of CvfA (Cho) Aim 3: Catalytic activity and structure of the HD domain of CvfA (Pérez-Alvarado)

Background and Significance: Conserved virulence factor A (CvfA) regulates genes in response to growth phase and nutrient availability in Streptococcus pyogenes [1]. Proper regulation of metabolic and virulence genes allows for adaptation to various host environments. Mild superficial infections, such as impetigo and pharyngitis, may develop into invasive diseases, such as necrotizing fasciitis and streptococcal toxic shock syndrome. CvfA comprises four domains: a transmembrane (TM) helix, a coiled-coil (CC) region, an RNA-binding K homology (KH) domain and a metal-dependent His-Asp-containing (HD) phosphohydrolase domain. Our working hypothesis is that CvfA selectively processes mRNA, utilizing the KH domain for RNA specificity and the HD domain for cleavage, resulting in either mRNA transcript depletion, or up-regulation by removal of repressor elements (Figure 1). Therapeutic CvfA inhibitors have the potential for high specificity against streptococcal diseases with limited adverse effects.

Research Strategy (Aims 1 and 3): The RNA specificity of the KH domain will be studied by in vitro selection and gel shift assays. This approach is expected to identify other mRNA transcripts targeted by CvfA. We have expanded our structural studies to include dual-domain constructs that comprise both KH and HD domains to explore coordination between RNA binding and catalytic activity. Proteins containing HD domains are associated with signal transduction and nucleic acid metabolism [2]. Primarily divalent metal ions are coordinated to the conserved aspartate and histidine residues, but the identity of the bound metal is variable [3-10]. Results of kinetic studies using non-specific substrates reveal both phosphodiesterase and phosphatase activity. Studies are ongoing to characterize the metal-dependence with RNA sequences derived from the 5' UTR of regulated genes. An integrative approach combining NMR spectroscopy, X-ray crystallography, and biophysical methods will be used to correlate structure and function. Project 2: Investigation of a candidate universal influenza vaccine targeting the HA head Collaborators: David Verhoeven (Iowa State University) and Brett Sponseller (Iowa State University) Current Funding: Merck Investigator Studies Program Review Committee (MISP-RC) October 2016 to September 2018

Specific Aims: Development of a universal influenza vaccine may obviate the need for seasonal influenza vaccines that include multiple and variable strains.

Aim 1: Identify epitopes in hemagglutinin recognized by antibodies (Lee, Verhoeven) Aim 2: Optimize the vaccine strategy to enhance responses (Verhoeven, Sponseller) Aim 3: Test the robustness of protection using ferrets (Verhoeven)

Background and Significance: A universal influenza vaccine requires epitopes that are protective across multiple subtypes and both phylogenic groups. An equine H3N8 based vaccine has been shown to elicit broadly neutralizing antibodies against H1N1 pnd09 and Brisbane H3N2 strains. Sequence analysis and homology modeling were used to map a conformational epitope in the head region of hemagglutinin (Figure 3).

Figure 1. CvfA bound to enolase. The KH domain targets mRNA for cleavage by the HD domain.

ENO

5‛

3‛

mRNA

✄

CvfA-enolase complex regulates expression of glycolytic and virulence genes in response to nutrient availability.

KH

HD

CC

TM

Figure 2. A potential conformational epitope from equine H3N8 HA.

Research – Brian Lee

2

Research Strategy: (Aim 1) Two parallel approaches will be used to identify conformational epitopes within hemagglutinin using Fab fragments derived from broadly neutralizing antibodies: a) crosslinking mass spectrometry, and b) structural studies by X-ray crystallography. The conformational epitopes will be verified by mutational analysis using recombinant hemagglutinin binding to Fab fragments assayed by ELISA. Surface plasmon resonance (Biacore-T100, GE) will assay the binding affinity and kinetics of hemagglutinin with matrix immobilized Fab fragments [11]. Project 3: Mechanism of antibiotic resistant Streptococcus pneumoniae using a novel lamb model of RSV co-infection Collaborators: David Verhoeven, Mark Ackermann and Edward Yu (Iowa State University) Current Funding: Merck Investigator Studies Program Review Committee (MISP-RC) November 2016 to October 2017

Specific Aims: Antimicrobial resistance in Streptococcus pneumonia is a serious threat to human health. Bacterial infections leading to pneumonia are often preceded by respiratory syncytial virus (RSV) infection.

Aim 1: Examine pathogenesis using a novel lamb model of co-infection (Verhoeven, Ackermann). Aim 2: Structural and functional studies of macrolide resistance from Mef(E) and Mel (Lee, Yu).

Background and Significance: In S. pneumoniae, macrolide efflux is associated with two genes, mef(E) and mel, regulated by transcriptional attenuation [12]. Both genes are found within moveable genetic elements in serovars associated with the M phenotype [13]. No structural studies have been reported for either Mef(E), the macrolide efflux protein, or Mel, the associated ATP binding cassette (ABC) protein.

Research Strategy (Aim 2): For structural studies of macrolide resistance, the mef(E) and mel genes from serotype 19A will be cloned and expressed. The membrane protein, Mef(E), will be assayed for extraction with various detergents [14]. For the ABC protein, Mel, two hypotheses will be tested (Figure 2) by crosslinking and pull-down assays. After purification, screens will assay crystallization conditions for X-ray diffraction studies. Structures will be solved by molecular replacement [15]. Bibliography 1. Kang SO, et al. Virulence gene regulation by CvfA, a putative RNase: the CvfA-enolase complex in Streptococcus pyogenes

links nutritional stress, growth-phase control, and virulence gene expression. Infect Immun. (2010) 78:2754-67. 2. Aravind L, Koonin EV. The HD domain defines a new superfamily of metal-dependent phosphohydrolases. Trends

Biochem Sci. (1998) 23:469-72. 3. Oganesyan V, et al. Structure of O67745_ AQUAE, a hypothetical protein from Aquifex aeolicus. Acta Crystallogr F.

(2007) 63:369-74. 4. Hogg T, et al. Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog

modulates (p)ppGpp metabolism during the stringent response [corrected]. Cell. (2004) 117:57-68. 5. Kondo N, et al. Structure of dNTP-inducible dNTP triphosphohydrolase: insight into broad specificity for dNTPs and

triphosphohydrolase-type hydrolysis. Acta Crystallogr D. (2007) 63:230-9. 6. Zimmerman MD, et al. Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-

domain phosphohydrolase: The 5'-deoxyribonucleotidase YfbR from Escherichia coli. J Mol Biol. (2008) 378:215-26. 7. 2PQ7, Crystal structure of predicted HD superfamily hydrolase (104161995) from uncultured Thermotogales bacterium at

1.45 A resolution. (2007). 8. 2OGI, Crystal structure of conserved hypothetical protein TIGR00488 (NP_688652.1) from Streptococcus agalactiae 2603 at

1.85 A resolution. (2007). 9. 3CCG, Crystal structure of predicted HD superfamily hydrolase involved in NAD metabolism (NP_347894.1) from

Clostridium acetobutylicum at 1.50 A resolution. (2008). 10. 2O08, Crystal structure of hypothetical protein (NP_242193.1) from Bacillus halodurans at 1.90 A resolution. (2006). 11. Hearty S, et al. Measuring antibody-antigen binding kinetics using surface plasmon resonance. Methods Mol Biol. (2012)

907:411-42. 12. Chancey ST, et al. Transcriptional attenuation controls macrolide inducible efflux and resistance in Streptococcus

pneumoniae and in other Gram-positive bacteria containing mef/mel(msr(D)) elements. Plos One. (2015) 10:e0116254. 13. Varaldo PE, et al. Genetic elements responsible for erythromycin resistance in streptococci. Antimicrob Agents Chemother.

(2009) 53:343-53. 14. Bolla JR, et al. Biomolecular membrane protein crystallization. Philos Mag (Abingdon). (2012) 92:2648-61. 15. Rossmann MG. The molecular replacement method. Acta Crystallogr A. (1990) 46:73-82.

Figure 3. Models of macrolide resistance. A. Mel interacts with Mef(E) to form an ABC/MFS transporter. B. Mef(E) acts independently and Mel rescues inactivated ribosomes.