Tying up the Loose Ends: A Mathematically Knotted Protein.

Knots have attracted scientists in mathematics, physics, biology, and engineering. Long flexible thin strings easily knot and tangle as experienced in our daily life. Similarly, long polymer chains inevitably tend to get trapped into knots. Little is known about their formation or function in proteins despite >1,000 knotted proteins identified in nature. However, these protein knots are not mathematical knots with their backbone polypeptide chains because of their open termini, and the presence of a "knot" depends on the algorithm used to create path closure. Furthermore, it is generally not possible to control the topology of the unfolded states of proteins, therefore making it challenging to characterize functional and physicochemical properties of knotting in any polymer. Covalently linking the amino and carboxyl termini of the deeply trefoil-knotted YibK from Pseudomonas aeruginosa allowed us to create the truly backbone knotted protein by enzymatic peptide ligation. Moreover, we produced and investigated backbone cyclized YibK without any knotted structure. Thus, we could directly probe the effect of the backbone knot and the decrease in conformational entropy on protein folding. The backbone cyclization did not perturb the native structure and its cofactor binding affinity, but it substantially increased the thermal stability and reduced the aggregation propensity. The enhanced stability of a backbone knotted YibK could be mainly originated from an increased ruggedness of its free energy landscape and the destabilization of the denatured state by backbone cyclization with little contribution from a knot structure. Despite the heterogeneity in the side-chain compositions, the chemically unfolded cyclized YibK exhibited several macroscopic physico-chemical attributes that agree with theoretical predictions derived from polymer physics.

The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms.

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues.

Heterogeneous dynamics in partially disordered proteins.

Importance of disordered protein regions is increasingly recognized in biology, but their characterization remains challenging due to the lack of suitable experimental and theoretical methods. NMR experiments can detect multiple timescale dynamics and structural details of disordered protein regions, but their detailed interpretation is often difficult. Here we combine protein backbone 15N spin relaxation data with molecular dynamics (MD) simulations to detect not only heterogeneous dynamics of large partially disordered proteins but also their conformational ensembles. We observed that the rotational dynamics of folded regions in partially disordered proteins is dominated by similar rigid body rotation as in globular proteins, thereby being largely independent of flexible disordered linkers. Disordered regions, on the other hand, exhibit complex rotational motions with multiple timescales below ∼30 ns which are difficult to detect from experimental data alone, but can be captured by MD simulations. Combining MD simulations and backbone 15N spin relaxation data, measured applying segmental isotopic labeling with salt-inducible split intein, we resolved the conformational ensemble and dynamics of partially disordered periplasmic domain of TonB protein from Helicobacter pylori containing 250 residues. To demonstrate the universality of our approach, it was applied also to the partially disordered region of chicken Engrailed 2. Our results pave the way in understanding how TonB transfers energy from inner membrane to the outer membrane receptors in Gram-negative bacteria, as well as the function of other proteins with disordered domains.

NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein.

The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.

The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from cis-splicing inteins.

Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 Å-resolution crystal structure of a fused variant from the naturally split gp41-1 intein, previously identified from environmental metagenomic sequence data. The structure of the 125-residue gp41-1 intein revealed a compact pseudo-C2-symmetry commonly found in the Hedgehog/Intein superfamily with extensive charge-charge interactions between the split N- and C-terminal intein fragments that are common among naturally occurring split inteins. We successfully created orthogonal split inteins by engineering a similar charge network into the same region of a cis-splicing intein. This strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins. DATABASE: Structural data are available in the RCSB Protein Data Bank under the accession number 6QAZ.

Biosynthesis of the Bis-Prenylated Alkaloids Muscoride A and B.

Prenylation is a common step in the biosynthesis of many natural products and plays an important role in increasing their structural diversity and enhancing biological activity. Muscoride A is a linear peptide alkaloid that contain two contiguous oxazoles and unusual prenyl groups that protect the amino- and carboxy-termini. Here we identified the 12.7 kb muscoride (mus) biosynthetic gene clusters from Nostoc spp. PCC 7906 and UHCC 0398. The mus biosynthetic gene clusters encode enzymes for the heterocyclization, oxidation, and prenylation of the MusE precursor protein. The mus biosynthetic gene clusters encode two copies of the cyanobactin prenyltransferase, MusF1 and MusF2. The predicted tetrapeptide substrate of MusF1 and MusF2 was synthesized through a novel tandem cyclization route in only eight steps. Biochemical assays demonstrated that MusF1 acts on the carboxy-terminus while MusF2 acts on the amino-terminus of the tetrapeptide substrate. We show that the MusF2 enzyme catalyzes the reverse or forward prenylation of amino-termini from Nostoc spp. PCC 7906 and UHCC 0398, respectively. This finding expands the regiospecific chemical functionality of cyanobactin prenyltransferases and the chemical diversity of the cyanobactin family of natural products to include bis-prenylated polyoxazole linear peptides.

Structure of the UspA1 protein fragment from Moraxella catarrhalis responsible for C3d binding.

The gram-negative bacterium Moraxella catarrhalis infects humans exclusively, causing various respiratory tract diseases, including acute otitis media in children, septicaemia or meningitis in adults, and pneumonia in the elderly. To do so, M. catarrhalis expresses virulence factors facilitating its entry and survival in the host. Among them are the ubiquitous surface proteins (Usps): A1, A2, and A2H, which all belong to the trimeric autotransporter adhesin family. They bind extracellular matrix molecules and inhibit the classical and alternative pathways of the complement cascade by recruiting complement regulators C3d and C4b binding protein. Here, we report the 2.5 Šresolution X-ray structure of UspA1299-452, which previous work had suggested contained the canonical C3d binding site found in both UspA1 and UspA2. We show that this fragment of the passenger domain contains part of the long neck domain (residues 299-336) and a fragment of the stalk (residues 337-452). The coiled-coil stalk is left-handed, with 7 polar residues from each chain facing the core and coordinating chloride ions or water molecules. Despite the previous reports of tight binding in serum-based assays, we were not able to demonstrate binding between C3d and UspA1299-452 using ELISA or biolayer interferometry, and the two proteins run separately on size-exclusion chromatography. Microscale thermophoresis suggested that the dissociation constant was 140.5 ±â€¯8.4 µM. We therefore suggest that full-length proteins or other additional factors are important in UspA1-C3d interactions. Other molecules on the bacterial surface or present in serum may enhance binding of those two molecules.

Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site.

Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.

Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation.

Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.

Crystal structure of a tripartite complex between C3dg, C-terminal domains of factor H and OspE of Borrelia burgdorferi.

Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19-20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the "common microbial binding site" on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.

Structural Basis for the Persistence of Homing Endonucleases in Transcription Factor IIB Inteins.

Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.

Segmental isotopic labeling of a single-domain globular protein without any refolding step by an asparaginyl endopeptidase.

Asparaginyl endopeptidases (AEPs) catalyze head-to-tail backbone cyclization of naturally occurring cyclic peptides such as cyclotides, and have become an important peptide-engineering tool for macrocyclization and peptide ligation. Here, we report efficient protein ligation in trans by mimicking efficient backbone cyclization by an AEP without any excess of reactants. We demonstrate a practical application of segmental isotopic labeling for NMR studies of a single-domain globular protein without any refolding step using the recombinant AEP prepared from Escherichia coli. This simple protein ligation approach using an AEP could be applied for incorporation of various biophysical probes into proteins as well as post-translational production of full-length proteins.

Yersinia infection tools-characterization of structure and function of adhesins.

Among the seventeen species of the Gram-negative genus Yersinia, three have been shown to be virulent and pathogenic to humans and animals-Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis. In order to be so, they are armoured with various factors that help them adhere to tissues and organelles, cross the cellular barrier and escape the immune system during host invasion. The group of proteins that mediate pathogen-host interactions constitute adhesins. Invasin, Ail, YadA, YadB, YadC, Pla, and pH 6 antigen belong to the most prominent and best-known Yersinia adhesins. They act at different times and stages of infection complementing each other by their ability to bind a variety of host molecules such as collagen, fibronectin, laminin, ß1 integrins, and complement regulators. All the proteins are anchored in the bacterial outer membrane (OM), often forming rod-like or fimbrial-like structures that protrude to the extracellular milieu. Structural studies have shown that the anchor region forms a ß-barrel composed of 8, 10, or 12 antiparallel ß-strands. Depending on the protein, the extracellular part can be composed of several domains belonging to the immunoglobulin fold superfamily, or form a coiled-coil structure with globular head domain at the end, or just constitute several loops connecting individual ß-strands in the ß-barrel. Those extracellular regions define the activity of each adhesin. This review focuses on the structure and function of these important molecules, and their role in pathogenesis.

The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation.

Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the ß-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the ß-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the ß-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not.