The N-terminal domain of RfaH plays an active role in protein fold-switching

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.     

N-terminal fragment of the anti-angiogenic human endostatin binds copper(II) with very high affinity

A histidine-rich peptide HSHRDFQPVLHL-NH₂ (L), identical with the N-terminal fragment of the anti-angiogenic human endostatin has been synthesized. Endostatin is a recently identified broad spectrum angiogenesis inhibitor, which inhibits 65 different tumor types. The N-terminal 25-mer peptide fragment of human endostatin has the same antitumor effect as the entire protein. The zinc(II) binding is crucial for the antitumor effect in both cases. Our peptide may provide all critical interactions for zinc(II) binding present in the N-terminal 25-mer peptide fragment. In addition, the N-terminus of human endostatin has a supposedly high affinity binding site for copper(II), similar to human serum albumin. Since copper(II) is intimately involved in angiogenesis, this may have biological relevance.In order to determine the metal binding properties of the N-terminal fragment of endostatin, we performed equilibrium, UV-visible (UV-vis), CD, EPR and NMR studies on the zinc(II) and copper(II) complexes of L. In the presence of zinc(II) the formation of a stable {NH₂, 3N_im, COO⁻} coordinated complex was detected in the neutral pH-range. This coordination mode is probably identical to that present in the zinc(II) complex of the above mentioned N-terminal 25-mer peptide fragment of human endostatin. Moreover, L has extremely high copper(II) binding affinity, close to those of copper-containing metalloenzymes, and forms albumin-like {NH₂, N⁻, N⁻, N_im} coordinated copper(II) complex in the neutral pH-range, which may suggest that copper(II) binding is involved in the biological activity of endostatin.     

The N-terminal domain of NPC1L1 protein binds cholesterol and plays essential roles in cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption.     

The N-terminal domain of the Schaaf–Yang syndrome protein MAGEL2 likely has a role in RNA metabolism

MAGEL2 encodes the L2 member of the melanoma-associated antigen gene (MAGE) protein family, truncating mutations of which can cause Schaaf-Yang syndrome, an autism spectrum disorder. MAGEL2 is also inactivated in Prader–Willi syndrome, which overlaps clinically and mechanistically with Schaaf–Yang syndrome. Studies to date have only investigated the C-terminal portion of the MAGEL2 protein, containing the MAGE homology domain that interacts with RING-E3 ubiquitin ligases and deubiquitinases to form protein complexes that modify protein ubiquitination. In contrast, the N-terminal portion of the MAGEL2 protein has never been studied. Here, we find that MAGEL2 has a low-complexity intrinsically disordered N-terminus rich in Pro-X_n-Gly motifs that is predicted to mediate liquid–liquid phase separation to form biomolecular condensates. We used proximity-dependent biotin identification (BioID) and liquid chromatography–tandem mass spectrometry to identify MAGEL2-proximal proteins, then clustered these proteins into functional networks. We determined that coding mutations analogous to disruptive mutations in other MAGE proteins alter these networks in biologically relevant ways. Proteins identified as proximal to the N-terminal portion of MAGEL2 are primarily involved in mRNA metabolic processes and include three mRNA N 6-methyladenosine (m⁶A)-binding YTHDF proteins and two RNA interference-mediating TNRC6 proteins. We found that YTHDF2 coimmunoprecipitates with MAGEL2, and coexpression of MAGEL2 reduces the nuclear accumulation of YTHDF2 after heat shock. We suggest that the N-terminal region of MAGEL2 may have a role in RNA metabolism and in particular the regulation of mRNAs modified by m⁶A methylation. These results provide mechanistic insight into pathogenic MAGEL2 mutations associated with Schaaf–Yang syndrome and related disorders.     

An autonomous N-terminal transactivation domain in Fos protein plays a crucial role in transformation.

To date, three functional domains have been defined in c-Fos and v-Fos proteins and have been shown to play a role in transactivation: the leucine zipper mediating hetero-dimerization, the basic DNA contact site, and a C-terminally located transactivation domain (C-TA) harbouring the HOB1 and HOB2 motifs. While the bZip region, consisting of the leucine zipper and the DNA contact site, is indispensable for transformation, the C-TA domain is not required and is actually altered by internal deletions in the FBR-MuSV. We now show that the N-terminal regions of c-Fos and v-Fos contain a second transactivation domain (N-TA). A functionally crucial motif within the N-TA domain, termed NTM, was pinpointed to a approximately 25 amino acid stretch around positions 60-84 which is highly conserved in FosB. Analysis of LexA fusion proteins showed that the N-TA domains of both c-Fos and FosB function in an autonomous fashion in both fibroblasts and yeast. Most importantly, deletion of the NTM motif impairs the transforming properties of v-Fos. Apart from the bZip region, the N-TA domain is the only functional domain required for transformation by v-Fos, at least when its expression is driven by the strong FBR-MuSV-LTR promoter.     

The N-terminal domain of 5-lipoxygenase binds calcium and mediates calcium stimulation of enzyme activity

Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidonic acid into leukotrienes and lipoxins, mediators and modulators of inflammation. In this study, we localized a stimulatory Ca(2+)-binding site to the N-terminal region of the enzyme. Thus, in a (45)Ca(2+) overlay assay, the N-terminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-transferase) bound radioactive calcium to about the same extent as intact 5-LO. The glutathione S-transferase fusion protein of the C-terminal part of 5-LO (amino acids 120-673) showed much weaker binding. A model of a putative 5-LO N-terminal domain was calculated based on the structure of rabbit reticulocyte 15-LO. This model resembles beta-sandwich C2 domains of other Ca(2+)-binding proteins. Comparison of our model with the C2 domain of cytosolic phospholipase A(2) suggested a number of amino acids, located in the loops that connect the beta-strands, as potential Ca(2+) ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca(2+) binding and a requirement for higher Ca(2+) concentrations to stimulate enzyme activity. Our data indicate that an N-terminal beta-sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity.     

The structure of the NPC1L1 N-terminal domain in a closed conformation

Background

NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels.

Principal Findings

Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent.

Conclusion

The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.     

The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR.

Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.     

The structure of the human centrin 2-xeroderma pigmentosum group C protein complex

Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered alpha-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an alpha-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.     

The N-terminal rhodanese domain from Azotobacter vinelandii has a stable and folded structure independently of the C-terminal domain

Sulfurtransferase are enzymes involved in the formation, conversion and transport of compounds containing sulfane-sulfur atoms. Although the three-dimensional structure of the rhodanese from the nitrogen-fixing bacterium Azotobacter vinelandii is known, the role of its two domains in the protein conformational stability is still obscure. We have evaluated the susceptibility to proteolytic degradation of the two domains of the enzyme. The two domains show different resistance to the endoproteinases and, in particular, the N-terminal domain shows to be more stable to digestion during time than the C-terminal one. Cloning and overexpression of the N-terminal domain of the protein was performed to better understand its functional and structural role. The recombinant N-terminal domain of rhodanese A. vinelandii is soluble in water solution and the spectroscopic studies by circular dichroism and heteronuclear NMR spectroscopy indicate a stable fold of the protein with the expected alpha/beta topology. The results indicate that this N-terminal domain has already got all the elements necessary for an C-terminal domain independent folding. Its solution structure by NMR, actually under course, will be a valid contribution to understand the role of this domain in the folding process of the sulfurtransferase.     

Xeroderma pigmentosum group C protein possesses a high affinity binding site to human centrin 2 and calmodulin

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca2+-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca2+ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway. Analysis of the XPC sequence (940 residues), using a calmodulin target recognition software, enabled us to predict two putative binding sites. The binding properties of the two corresponding peptides were investigated by isothermal titration calorimetry. Only one of the peptides (P1-XPC) interacts strongly (Ka = 2.2 x 10(8) m-1, stoichiometry 1:1) with HsCen2 in a Ca2+-dependent manner. This peptide also binds, with a similar affinity (Ka = 1.1 x 10(8) m-1) to a C-terminal construct of HsCen2, indicating that the interaction with the integral protein is mainly the result of the contribution of the C-terminal half. The second peptide (P2-XPC) failed to show any detectable binding either to HsCen2 or to its C-terminal lobe. The two peptides interact with different affinities and mechanisms with calmodulin. Circular dichroism and nuclear magnetic resonance were used to structurally characterize the complex formed by the C-terminal domain of HsCen2 with P1-XPC.     

The N-terminal domain influences the structure and property of protein phosphatase 1

Molecular and Cellular Biochemistry - The protein phosphatase 1 has conserved cores in PPP gene family flanked by non-conserved N-terminal domains. PP1 with residues 1–8 deleted or...     

The low affinity binding sites of the vitamin D-dependent calcium-binding protein and their functional role in calcium transport

Calcium- and other divalent cation-binding properties of the vitamin D-dependent calcium-binding protein isolated from the duodenal mucosa of chicks were studied using the flow dialysis technique and ⁴⁵Ca. It was found that the calcium-binding protein along with the high affinity binding sites has approximately 40 low affinity binding sites with K_a of about 1000 M^?1. The low affinity sites possess of certain specificity towards binding of Ca. The affinity of the calcium-bindin protein for other divalent cations depends on the ionic radius. It is suggested that the low affinity binding sites of the calcium-binding protein take part in calcium transport organization across the intestinal epithelium.     

The N-terminal domain of Bcl-xL reversibly binds membranes in a pH-dependent manner

Bcl-xL regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-xLDeltaTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bcl-xLDeltaTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-xL activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-xL mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis.     

The proline-rich domain of tau plays a role in interactions with actin

Background  

The microtubule-associated protein tau is able to interact with actin and serves as a cross-linker between the microtubule and actin networks. The microtubule-binding domain of tau is known to be involved in its interaction with actin. Here, we address the question of whether the other domains of tau also interact with actin.     

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.     

The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner

Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.