Special - October 2010
Short description: Visualizing molecules linked to disease can give insight into how to strike back. All the more reason for aiming to solve structures of these proteins.
Just as a map of the terrain can help a general strategically plan an attack, understanding the molecular landscape underlying a disease can help plan where and how to strike back. A number of recent papers describe structures that give insight into the functioning of pathogens, elucidate the cellular processes that they target during infection, or visualize inhibitors that can prevent or ameliorate disease.
Understanding unusual or species-specific processes can also provide avenues for attacking a pathogen that do not impinge upon host cell function. Some bacteria have unusual mechanisms for finding nutrients when they are scarce, and these include mechanisms for scavenging iron and for heme uptake. Ferric iron (Fe3+) can be difficult for aerobic bacteria to get hold of and some species, including certain pathogens, have developed secreted molecules known as siderophores that chelate iron and are then taken up via ABC transporters. The siderophore petrobactin, a catecholate, is produced by the Bacillus cereus family, which includes the pathogen B. anthracis, and is associated with virulence. It chelates ferric iron through a rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA), which enables petrobactin and its cargo of iron to escape sequestration by the mammalian immune protein siderocalin.
Sherman and colleagues (PSI MCSG) have determined the structure of a key enzyme in petrobactin synthesis, AsbF. AsbF is a dehydratase that converts 3-dehydroshikimate to 3,4-DHBA 1 . The structure was obtained in a complex with 3,4-DHBA and manganese ion and provides insight into the catalysis of the petrobactin precursor. The MCSG team then addressed the question of how petrobactin was transported when complexed with iron. The YclQ protein in Bacillus species was already known to be a transporter for petrobactin. Raymond and colleagues (PSI MCSG) now provide evidence that YclQ specifically binds iron-chelated petrobactin for uptake and have determined its structure at 1.75 Å (ref. 2). YclQ consists of amino- and carboxy-terminal α/β domains with a ligand-binding cleft between the domains and structurally resembles the catecholate-binding protein CeuE. This work may lead to greater understanding of the siderophore uptake mechanism in B. anthracis. Looking at another mechanism for iron uptake, Phillips and colleagues (PSI CESG) have examined heme uptake in Streptococcus pyogenes 3 , which involves direct heme transfer by the heme-containing protein Shp to HtsA, a lipoprotein component of an ABC transporter. The 2.1 Å crystal structure of the Shp heme-binding domain complexed to heme reveals a unique binding mode, with the heme-iron coordinated by two methionines.
Bacteria-specific pathways of nucleotide metabolism have also recently been explored through structural analyses, providing distinct examples of pathogen-specific factors that could be possible drug targets. Kohen and colleagues (PSI JCSG) have examined ThyX, which is a bacterial flavin-dependent thymidylate synthase (FDTS). The crystal structure of the ThyX protein 4 , together with isotope substitution to follow the path of the proton during catalysis, suggests that ThyX uses a very different mechanism and a different cofactor for synthesizing thymidylate compared with those used by mammalian thymidylate synthetases. In the bacterial FDTS, the serine nucleophile is dispensable, and the authors argue that the reaction consists of hydride transfer to a uracil ring followed by isomerization of the resulting intermediate. Such a mechanism has not been reported previously for thymine biosynthesis or for other nucleotide methylations, thus providing an Achilles heel to combat bacterial pathogens that use this pathway.
The structures of bacterial virulence factors have been examined by the JCSG in collaboration with Kazuhisa Sekimizu's group at the University of Tokyo in Japan, who have utilized a silkworm infection model to identify the conserved genes cvfA, cvfB, cvfC and sarZ as Staphylococcus aureus virulence factors. The collaboration has now characterized the CvfB protein from S. aureus, showing it to be an RNA-binding protein capable of specifically binding poly(U), (A) or (G) in vitro via its WH domain 5 . The authors put 25 orthologs of CvfB through the high-throughput pipeline at PSI JCSG, eventually solving the crystal structure of the Streptococcus pneumoniae homolog, which turns out to have three S1 domains and a WH domain arranged in an L shape. The third S1 domain, S1C, looks like an RNA-binding domain, structurally resembling domain 1 of the archaeal translation initiation factor 1α. The positive electrostatic surface on S1C is conserved, contributing to an extended RNA-binding region connected to the WH domain. Binding experiments suggest that these two domains contribute most to RNA interaction. Deletion of CvfB leads to reduced hemolytic activity, which may be a distinct phenotype, with the WH domain able to rescue this phenotype and so implicating this protein in a specific process contributing to the pathogenicity.
Bacterial antibiotic resistance can involve specialized mechanisms for transporting the antibiotic across cell membranes, and Burley and colleagues (PSI NYSGXRC) have determined the structure of a BenF-like porin integral membrane protein 6 .
A better comprehension of the mode of pathogen attack on its host is another contribution that structural analyses can make toward therapy. Montelione and colleagues (PSI NESG) have examined the NS1 protein, an influenza A virulence factor that interferes with host-cell mRNA processing. The authors have determined the structure of the NS1A effector domain, the domain responsible for the interference, in complex with zinc fingers from the human cellular polyadenylation and specificity factor CPSF30, an mRNA-processing factor 7 . The complex has an unusual tetrameric structure, with the NS1A domains arranged head to head and the CPSF30 Zn-finger domains wrapped around them. The binding pocket on NS1A is hydrophobic and mutagenesis identified residues required for interaction with CPSF30. Genetically engineered influenza A viruses with mutations in the CPSF30-binding pocket of their NS1 proteins have attenuated virulence. By binding to CPSF30, the influenza NS1 protein suppresses host-cell production of interferon-β, a protein produced in the innate immune response that activates apoptosis of infected cells; blocking the NS1–CPSF30 interaction thus results in an attenuated viral infection.
Viral proteins can affect a variety of cellular pathways. Markley and colleagues (PSI CESG) have determined the structure of the leader protein in mengovirus 8 , a rodent virus that can cause an acute fever in humans. The leader protein affects host nucleocytoplasmic transport and turns out to have an unusual zinc-finger structure that may enable further elucidation of its function and targets.
Whereas the structures of factors involved in pathogenesis may give clues that drive the discovery of novel drugs, the structures of complexes with known inhibitors can lead to the design of more effective drugs. The microsporidian Enterocytozoon bieneusi is a pathogen in immunosuppressed patients. Weiss and colleagues (PSI NYSGXRC) have determined the structure of fumagillin, an inhibitor of methionine aminopeptidase (MetAp2), in complex with the structure of a MetAP2 homolog 9 . This structure was used for homology modeling of the structure of E. bieneusi MetAP2, indicating the utility of solving a homologous structure for potentially generating ways of designing better inhibitors to target a pathogen.
Targeting endogenous processes that have gone awry is key to tackling cancer and other diseases. Onconase (ONC) is an inhibitor derived from the frog Rana pipiens and is in advanced clinical trials as an anti-tumor agent. It is an RNase homolog and its crystal structure in a complex with nucleic acid has been determined at 1.65 Å by Raines and colleagues (PSI CESG) 10 . The structure indicates the basis of its specificity, explaining how ONC differentiates between adenine and guanine. Glu 91 of ONC forms two hydrogen bonds with the guanine base in d(AUGA) and the structural analysis guided rational mutagenesis to shift the binding preference toward either A or G. Another example is the surprising new insight gleaned from the structure of the enzyme aspartoacyclase, defects in which cause Canavan's disease, where inhibition of the breakdown of N-aspartyl aspartate affects development of nervous tissue. This new structure, also from the CESG, reveals the connections between genetic lesions and the inability of the protein to function 11 .
Investigating the possible use of bacterial products as cancer therapeutics, Thorson and colleagues (PSI CESG) have examined the self-sacrifice mechanism of drug resistance by the bacterial protein CalC, which binds the antibiotic calicheamicin, which has an enediyne 'warhead' that breaks DNA. Upon enediyne binding, CalC breaks down, taking out the enediyne in the process. The crystal structure of CalC shows that it is a member of the START family 12 . CalC also contains a potential DNA-binding domain, which the authors suggest may help bring it to the site of enediyne attack. The differing chemical groups added to the enediynes in diferent species, and which give them the ability to selectively target other organisms' DNA, is controlled by various enzymes whose structures have been determined by Phillips and colleagues at PSI CESG to reveal novel ways of creating new potential drug molecules 13 .
Human disease can also arise from aberrant inhibition of our own proteins. The eukaryotic pyrimidine 5′-nucleotidase type 1 (P5N-1) catalyzes the dephosphorylation of pyrimidine 5′-mononucleotides during RNA catabolism. P5N-1 deficiency, caused either by mutation or as a result of lead poisoning, leads to non-spherocytic hemolytic anemia. Phillips and colleagues (PSI CESG) have determined the crystal structure of mouse P5N-1 at 2.35 Å resolution, and find that the fold resembles that of members of the haloacid dehydrogenase superfamily 14 . Using structures representing different states in the reaction cycle, the authors provide evidence for a reaction mechanism. In addition, the structure in complex with Pb(II) shows that it resides in the active site, but away from the cationic cavity, binding in a manner distinct from the magnesium observed in the uninhibited enzyme and indicating a likely basis for lead poisoning of this enzyme and a contributing factor to lead-related illnesses.
Recent papers also examine endogenous proteins implicated in disease. RPM-1 (regulator of presynaptic morphology 1) is involved in ubiquitination in the nervous system and has been linked to neural development. The structure of the first and second PHR domains have now been determined by Burley and colleagues (PSI NYSGXRC), providing insights into the link between Ub-mediated turnover and neurodegeneration 15 . As another example of this approach, indoleamine 2,3-dioxygenase (IDO) has been linked to some pathological conditions related to immune system function including immune evasion by cancers. The TDO complex, related functionally to IDO, has been determined in the reduced Fe(II) bound state, in complexes with L-Trp or 6-fluoro-Trp, by Tong and colleagues (PSI NESG) 16 . The structures of these complexes not only reveal the basis of stereospecificity, but also suggest that TDO is an induced fit enzyme, with a disordered region forming a wall that closes off the active site upon substrate binding. The active site in a second monomer in the asymmetric unit differs in that the substrate Trp side chain is not as deeply bound and the loop is still disordered, which may prove to represent an intermediate stage in the binding mechanism.
These representative examples from the PSI reveal a variety of approaches for examining the structure and mechanism of microbial and cellular factors that are related to disease. Unexpected and unusual mechanisms have, indeed, been identified, which in turn suggests potential new approaches for striking back at disease. Such vignettes also provide examples of successful interactions and outreach with the community on protein structures that have been determined in the PSI that are highly relevant to disease.
B. F. Pfleger, Y. Kim, T. D. Nusca, N. Maltseva, J. Y. Lee et al. Structural and functional analysis of AsbF: origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis.
Proc. Natl Acad. Sci. USA 105, 17133-17138 (2008). doi:10.1073/pnas.0808118105
A. M. Zawadzka, Y. Kim, N. Maltseva, R. Nichiporuk, Y. Fan et al. Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore.
Proc. Natl Acad. Sci. USA 106, 21854-21859 (2009). doi:10.1073/pnas.0904793106
R. Aranda, 4th, C. E. Worley, M. Liu, E. Bitto, M. S. Cates. Bis-methionyl coordination in the crystal structure of the heme-binding domain of the streptococcal cell surface protein Shp.
J. Mol. Biol. 374, 374-83 (2007). doi:10.1016/j.jmb.2007.08.058
E. M. Koehn, T. Fleischmann, J. A. Conrad, B. A. Palfey, S. A. Lesley et al. An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene.
Nature 458, 919-23 (2009). doi:10.1038/nature07973
Y. Matsumoto, Q. Xu, S. Miyazaki, C. Kaito, C. L. Farr et al. Structure of a virulence regulatory factor CvfB reveals a novel winged-helix RNA binding module.
Structure 8, 537-547 (2010). doi:10.1016/j.str.2010.02.007
P. Sampathkumar, F. Lu, X. Zhao, Z. Li, J. Gilmore et al. Structure of a putative BenF-like porin from Pseudomonas fluorescens Pf-5 at 2.6 Å resolution.
Proteins (2010). doi:10.1002/prot.22829
K. Das, L.-C. Ma, R. Xiao, B. Radvansky, J. Aramini et al. Structural basis for suppression by influenza A virus of a host antiviral response.
Proc. Natl Acad. Sci. USA 105, 13092-13097 (2008). doi:10.1073/pnas.0805213105
C. C. Cornilescu, F. W. Porter, K. Q. Zhao, A. C. Palmenberg & J. L. Markley. NMR structure of the mengovirus leader protein zinc-finger domain.
FEBS Lett. 582, 896-900 (2008). doi:10.1016/j.febslet.2008.02.023
J. J. Alvarado, A. Nemkal, J. M. Sauder, M. Russell, D. E. Akiyoshi et al. Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470.
Mol. Biochem. Parasitol. 168, 158-167 (2009). doi:10.1016/j.molbiopara.2009.07.008
J. E. Lee, E. Bae, C. A. Bingman, G. N. Phillips, Jr & R. T. Raines. Structural basis for catalysis by onconase.
J. Mol. Biol. 375, 165-177 (2008). doi:10.1016/j.jmb.2007.09.089
E. Bitto, C. A. Bingman, G. E. Wesenberg, J. G. McCoy & G. N. Phillips, Jr. Structure of aspartoacylase, the brain enzyme impaired in Canavan disease.
Proc. Natl Acad. Sci. USA 104, 456-461 (2007). doi:10.1073/pnas.0607817104
S. Singh, M. H. Hager, C. Zhang, B. R. Griffith, M. S. Lee et al. Structural insight into the self-sacrifice mechanism of enediyne resistance.
ACS Chem. Biol. 1, 451-460 (2006). doi:10.1021/cb6002898
C. Zhang, E. Bitto, R. D. Goff, S. Singh, C. A. Bingman et al. Biochemical and structural insights of the early glycosylation steps in calicheamicin biosynthesis.
Chem. Biol. 15, 842-853 (2008). doi:10.1016/j.chembiol.2008.06.011
E. Bitto, C. A. Bingman, G. E. Wesenberg, J. G. McCoy & G. N. Phillips, Jr. Structure of pyrimidine 5′-nucleotidase type 1: Insight into mechanism of action and inhibition during lead poisoning.
J. Biol. Chem. 281, 20521-20529 (2006). doi:10.1074/jbc.M602000200
P. Sampathkumar, S. A. Ozyurt, S. A. Miller, K. T. Bain, M. E. Rutter et al. Structures of PHR domains from Mus musculus Phr1 (Mycbp2) explain the loss-of-function mutation (Gly1092 ← Glu) of the C. elegans ortholog RPM-1.
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F. Forouhar, J. L. Anderson, C. G. Mowat, S. M. Vorobiev, A. Hussain et al. Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase.
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