C. Martin Lawrence

CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea

Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.

Structural and Functional Studies of Archaeal Viruses

Viruses populate virtually every ecosystem on the planet, including the extreme acidic, thermal, and saline environments where archaeal organisms can dominate. For example, recent studies have identified crenarchaeal viruses in the hot springs of Yellowstone National Park and other high temperature environments worldwide. These viruses are often morphologically and genetically unique, with genomes that show little similarity to genes of known function, complicating efforts to understand their viral life cycles. Here, we review progress in understanding these fascinating viruses at the molecular level and the evolutionary insights coming from these studies.

Structure of the Fab Fragment of F105, a Broadly Reactive Anti-Human Immunodeficiency Virus (HIV) Antibody That Recognizes the CD4 Binding Site of HIV Type 1 gp120

ABSTRACT We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe43 of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.

The Structure of the CRISPR-Associated Protein Csa3 Provides Insight Into the Regulation of the CRISPR/Cas System

Adaptive immune systems have recently been recognized in prokaryotic organisms where, in response to viral infection, they incorporate short fragments of invader-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). In subsequent infections, the CRISPR loci are transcribed and processed into guide sequences for the neutralization of the invading RNA or DNA. The CRISPR-associated protein machinery (Cas) lies at the heart of this process, yet many of the molecular details of the CRISPR/Cas system remain to be elucidated. Here, we report the first structure of Csa3, a CRISPR-associated protein from Sulfolobus solfataricus (Sso1445), which reveals a dimeric two-domain protein. The N-terminal domain is a unique variation on the dinucleotide binding domain that orchestrates dimer formation. In addition, it utilizes two conserved sequence motifs [Thr-h-Gly-Phe-(Asn/Asp)-Glu-X(4)-Arg and Leu-X(2)-Gly-h-Arg] to construct a 2-fold symmetric pocket on the dimer axis. This pocket is likely to represent a regulatory ligand-binding site. The N-terminal domain is fused to a C-terminal MarR-like winged helix-turn-helix domain that is expected to be involved in DNA recognition. Overall, the unique domain architecture of Csa3 suggests a transcriptional regulator under allosteric control of the N-terminal domain. Alternatively, Csa3 may function in a larger complex, with the conserved cleft participating in protein-protein or protein-nucleic acid interactions. A similar N-terminal domain is also identified in Csx1, a second CRISPR-associated protein family of unknown function.

Something Old, Something New, Something Borrowed; How the Thermoacidophilic Archaeon Sulfolobus solfataricus Responds to Oxidative Stress

To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H2O2) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 µM H2O2. Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had significant changes in abundance. A recently characterized ferritin-like antioxidant protein, DPSL, was the most highly regulated species of mRNA and protein, in addition to being post-translationally modified. As expected, a number of antioxidant related mRNAs and proteins were differentially regulated. Three of these, DPSL, superoxide dismutase, and peroxiredoxin were shown to interact and likely form a novel supramolecular complex for mitigating oxidative damage. A scheme for the ability of this complex to perform multi-step reactions is presented. Despite the central role played by DPSL, cells maintained a lower level of protection after disruption of the dpsl gene, indicating a level of redundancy in the oxidative stress pathways of S. solfataricus. This work provides the first “omics” scale assessment of the oxidative stress response for an archeal organism and together with a network analysis using data from previous studies on bacteria and eukaryotes reveals evolutionarily conserved pathways where complex and overlapping defense mechanisms protect against oxygen toxicity.

Structure of the membrane proximal oxidoreductase domain of human Steap3, the dominant ferrireductase of the erythroid transferrin cycle.

The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor-mediated endocytosis. Upon endosomal acidification, iron is released from Tf, reduced to Fe2+ by Steap3, and transported across the endosomal membrane by divalent metal iron transporter 1. Steap3, the major ferrireductase in erythrocyte endosomes, is a member of a unique family of reductases. Steap3 is comprised of an N-terminal cytosolic oxidoreductase domain and a C-terminal heme-containing transmembrane domain. Cytosolic NADPH and a flavin are predicted cofactors, but the NADPH/flavin binding domain differs significantly from those in other eukaryotic reductases. Instead, Steap3 shows remarkable, although limited homology to FNO, an archaeal oxidoreductase. We have determined the crystal structure of the human Steap3 oxidoreductase domain in the absence and presence of NADPH. The structure reveals an FNO-like domain with an unexpected dimer interface and substrate binding sites that are well positioned to direct electron transfer from the cytosol to a heme moiety predicted to be fixed within the transmembrane domain. Here, we discuss possible gating mechanisms for electron transfer across the endosomal membrane.

Structure of D-63 from Sulfolobus Spindle-Shaped Virus 1: Surface Properties of the Dimeric Four-Helix Bundle Suggest an Adaptor Protein Function

ABSTRACT Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH ≤ 4.0) hot springs (≥70°C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly.

Cysteine usage in Sulfolobus spindle-shaped virus 1 and extension to hyperthermophilic viruses in general

Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs worldwide. The type virus, Sulfolobus spindle-shaped virus 1 (SSV1), has a double-stranded DNA genome that contains 34 open reading frames (ORFs). Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, tertiary protein structure can provide insight into protein function. We have thus undertaken a systematic investigation of the SSV1 proteome and report here on the F112 gene product. Biochemical, proteomic and structural studies reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Notably, the structure contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses, and reveals decreased cysteine content in the membrane proteins of hyperthermophilic viruses infecting Sulfolobales. The evolutionary implications of the SSV1 distribution are discussed.

Fossil record of an archaeal HK97-like provirus.

One of the outstanding questions in biology today is the origin of viruses. We have discovered a protein in the hyperthermophile Sulfolobus solfataricus while following proteome regulation during viral infection that led to the discovery of a fossil provirus. Characterization of the wild type and recombinant protein revealed that it assembled into virus-like particles with a diameter of ~32nm. Sequence and structural analyses showed that the likely proviral capsid protein, Sso2749, is homologous to a protein from Pyrococcus furiosus that forms virus-like particles using the HK-97 major capsid protein fold. The SsP2-provirus appears mosaic and contains proteins with similarity to, among others, eukaryotic herpesviruses and tailed dsDNA bacteriophage families, reinforcing the hypothesis of a common ancestral gene pool across all three domains of life. This is the first description of the HK-97 fold in a crenarchaeal virus and the first direct genomic connection of linocin-like protein cages to a virus.

Characterization of a Single b-type Heme, FAD, and Metal Binding Sites in the Transmembrane Domain of Six-transmembrane Epithelial Antigen of the Prostate (STEAP) Family Proteins

Background: Steap metalloreductases are critical to metal homeostasis and linked to multiple diseases. Results: Single b-type heme, FAD, and iron binding sites are identified in the Steap3 transmembrane domain and kinetically characterized. Conclusion: Steap transmembrane domains contain a single b-type heme and a high affinity FAD binding site that coordinates intrasubunit, transmembrane electron transfer. Significance: The findings extend to Steap family proteins in general, including Steap1. Six-transmembrane epithelial antigen of the prostate 3 (Steap3) is the major ferric reductase in developing erythrocytes. Steap family proteins are defined by a shared transmembrane domain that in Steap3 has been shown to function as a transmembrane electron shuttle, moving cytoplasmic electrons derived from NADPH across the lipid bilayer to the extracellular face where they are used to reduce Fe3+ to Fe2+ and potentially Cu2+ to Cu1+. Although the cytoplasmic N-terminal oxidoreductase domain of Steap3 and Steap4 are relatively well characterized, little work has been done to characterize the transmembrane domain of any member of the Steap family. Here we identify high affinity FAD and iron biding sites and characterize a single b-type heme binding site in the Steap3 transmembrane domain. Furthermore, we show that Steap3 is functional as a homodimer and that it utilizes an intrasubunit electron transfer pathway through the single heme moiety rather than an intersubunit electron pathway through a potential domain-swapped dimer. Importantly, the sequence motifs in the transmembrane domain that are associated with the FAD and metal binding sites are not only present in Steap2 and Steap4 but also in Steap1, which lacks the N-terminal oxidoreductase domain. This strongly suggests that Steap1 harbors latent oxidoreductase activity.