Cluster Of Hydrophobic Residues Research Articles

Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

A coarse-grained molecular dynamics investigation on spontaneous binding of Aβ1–40 fibrils with cholesterol-mixed DPPC bilayers

Alzheimer's disease is the most common form of dementia. Its aetiology is characterized by the misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet-rich Aβ oligomers/fibrils. Although multiple experimental studies have suggested that Aβ oligomers/fibrils interact with the cell membranes and perturb their structures and dynamics, the molecular mechanism of this interaction is still not fully understood. In the present work, we have performed a total of 120 μs-long simulations to investigate the interaction between trimeric or hexameric Aβ1–40 fibrils with either a 100% DPPC bilayer, a 70% DPPC-30% cholesterol bilayer or a 50% DPPC-50% cholesterol bilayer. Our simulation data capture the spontaneous binding of the aqueous Aβ1–40 fibrils with the membranes and show that the central hydrophobic amino acid cluster, the lysine residue adjacent to it and the C-terminal hydrophobic residues are all involved in the process. Moreover, our data show that while the Aβ1–40 fibril does not bind to the 100% DPPC bilayer, its binding affinity for the membrane increases with the amount of cholesterol. Overall, our data suggest that two clusters of hydrophobic residues and one lysine help Aβ1–40 fibrils establish stable interactions with a cholesterol-rich DPPC bilayer. These residues are likely to represent potential target regions for the design of inhibitors, thus opening new avenues in structure-based drug design against Aβ oligomer/fibril-membrane interaction.

Directed mutational scanning reveals a balance between acidic and hydrophobic residues in strong human activation domains.

SUMMARYAcidic activation domains are intrinsically disordered regions of the transcription factors that bind coactivators. The intrinsic disorder and low evolutionary conservation of activation domains have made it difficult to identify the sequence features that control activity. To address this problem, we designed thousands of variants in seven acidic activation domains and measured their activities with a high-throughput assay in human cell culture. We found that strong activation domain activity requires a balance between the number of acidic residues and aromatic and leucine residues. These findings motivated a predictor of acidic activation domains that scans the human proteome for clusters of aromatic and leucine residues embedded in regions of high acidity. This predictor identifies known activation domains and accurately predicts previously unidentified ones. Our results support a flexible acidic exposure model of activation domains in which the acidic residues solubilize hydrophobic motifs so that they can interact with coactivators. A record of this paper’s transparent peer review process is included in the supplemental information.

C-terminal troponin-I residues trap tropomyosin in the muscle thin filament blocked-state

Tropomyosin and troponin regulate muscle contraction by participating in a macromolecular scale steric-mechanism to control myosin-crossbridge – actin interactions and consequently contraction. At low-Ca2+, the C-terminal 30% of troponin subunit-I (TnI) is proposed to trap tropomyosin in a position on thin filaments that sterically interferes with myosin-binding, thus causing muscle relaxation. In contrast, at high-Ca2+, inhibition is released after the C-terminal domains dissociate from F-actin-tropomyosin as its component switch-peptide domain binds to the N-lobe of troponin-C (TnC). Recent, paradigm-shifting, cryo-EM reconstructions by the Namba group have revealed density attributed to TnI along cardiac muscle thin filaments at both low- and high-Ca2+ concentration. Modeling the reconstructions showed expected high-Ca2+ hydrophobic interactions of the TnI switch-peptide and TnC. However, under low-Ca2+ conditions, sparse interactions of TnI and tropomyosin, and in particular juxtaposition of non-polar switch-peptide residues and charged tropomyosin amino acids in the published model seem difficult to reconcile with an expected steric-blocking conformation. This anomaly is likely due to inaccurate fitting of tropomyosin into the cryo-EM volume. In the current study, the low-Ca2+ cryo-EM volume was fitted with a more accurate tropomyosin model and representation of cardiac TnI. Our results show that at low-Ca2+ a cluster of hydrophobic residues at the TnI switch-peptide and adjacent H4 helix (Ala149, Ala151, Met 154, Leu159, Gly160, Ala161, Ala163, Leu167, Leu169, Ala171, Leu173) draw-in tropomyosin surface residues (Ile143, Ile146, Ala151, Ile154), presumably attracting the entire tropomyosin cable to its myosin-blocking position on actin. The modeling confirms that neighboring TnI “inhibitory domain” residues (Arg145, Arg148) bind to thin filaments at actin residue Asp25, as previously suggested. ClusPro docking of TnI residues 137–184 to actin-tropomyosin, including the TnI inhibitory-domain, switch-peptide and Helix H4, verified the modeled configuration. Our residue-to-residue contact-mapping of the TnI-tropomyosin association lends itself to experimental validation and functional localization of disease-bearing mutations.

Development of an Antimicrobial Peptide-based Biosensor for the Monitoring of Bacterial Contaminations

Nisin is a cationic amphiphilic peptide consisting of 34 amino acids with a cluster of hydrophobic residues at the N-terminus and hydrophilic residues at the C-terminus. The mechanism of its action is based on its ability to attach to the bacteria cell membrane before to then cause cell lysis. This antimicrobial peptide was used as biological molecule for the development of a novel impedimetric label-free biosensor for the monitoring of bacterial contamination. The binding affinities of Nisin immobilized with its N-terminus and C-terminus were compared, highlighting the capability of the last configuration to obtain the best analytical performance of the developed biosensor when non-pathogenic Escherichia Coli O157:H7 and Listeria innocua cells were investigated.

A Shift in Aggregation Avoidance Strategy Marks a Long-Term Direction to Protein Evolution.

To detect a direction to evolution, without the pitfalls of reconstructing ancestral states, we need to compare "more evolved" to "less evolved" entities. But because all extant species have the same common ancestor, none are chronologically more evolved than any other. However, different gene families were born at different times, allowing us to compare young protein-coding genes to those that are older and hence have been evolving for longer. To be retained during evolution, a protein must not only have a function, but must also avoid toxic dysfunction such as protein aggregation. There is conflict between the two requirements: hydrophobic amino acids form the cores of protein folds, but also promote aggregation. Young genes avoid strongly hydrophobic amino acids, which is presumably the simplest solution to the aggregation problem. Here we show that young genes' few hydrophobic residues are clustered near one another along the primary sequence, presumably to assist folding. The higher aggregation risk created by the higher hydrophobicity of older genes is counteracted by more subtle effects in the ordering of the amino acids, including a reduction in the clustering of hydrophobic residues until they eventually become more interspersed than if distributed randomly. This interspersion has previously been reported to be a general property of proteins, but here we find that it is restricted to old genes. Quantitatively, the index of dispersion delineates a gradual trend, i.e., a decrease in the clustering of hydrophobic amino acids over billions of years.

Crystal structure of lipid A disaccharide synthase LpxB from Escherichia coli

Most Gram-negative bacteria are surrounded by a glycolipid called lipopolysaccharide (LPS), which forms a barrier to hydrophobic toxins and, in pathogenic bacteria, is a virulence factor. During LPS biosynthesis, a membrane-associated glycosyltransferase (LpxB) forms a tetra-acylated disaccharide that is further acylated to form the membrane anchor moiety of LPS. Here we solve the structure of a soluble and catalytically competent LpxB by X-ray crystallography. The structure reveals that LpxB has a glycosyltransferase-B family fold but with a highly intertwined, C-terminally swapped dimer comprising four domains. We identify key catalytic residues with a product, UDP, bound in the active site, as well as clusters of hydrophobic residues that likely mediate productive membrane association or capture of lipidic substrates. These studies provide the basis for rational design of antibiotics targeting a crucial step in LPS biosynthesis.

Microfluidic Turbulent Mixers, Time Resolved SAXS and Folding Intermediates of CheY

The process of folding a polypeptide chain from an unstructured random coil in denaturant to a functional 3 dimensional form is a rapid process that is perhaps biased very early on during the initial steps, even as the solvent conditions are transitioning from favoring denaturation to favoring folding. Access to kinetics in the microseconds to milliseconds time scale by multiple spectroscopic probes is crucial to interpreting these initial steps of folding. Time resolved small angle X-ray scattering (trSAXS) is a powerful technique for studying 3 dimensional shapes of proteins as they transition from unfolded to native structures. By interfacing microfluidic devices with powerful x-ray sources we can access timescales in the sub-hundred microseconds range. Structural details determined using trSAXS of the kinetic refolding process of CheY reveal a burst phase in the sub 50-microsecond time-scale, that is perhaps structured around a local-in-sequence cluster of hydrophobic residues.

NMR structure and conformational dynamics of AtPDFL2.1, a defensin-like peptide from Arabidopsis thaliana

Plant defensins constitute the innate immune response against pathogens such as fungi and bacteria. Typical plant defensins are small, basic peptides that possess a characteristic three-dimensional fold stabilized by three or four disulfide bridges. In addition to known defensin genes, the Arabidopsis genome comprises >300 defensin-like genes coding for small cysteine-rich peptides. One of such genes encodes for AtPDFL2.1, a putative antifungal peptide of 55 amino acids, with six cysteine residues in its primary sequence. To understand the functional role of AtPDFL2.1, we carried out antifungal activity assays and determined its high-resolution three-dimensional structure using multidimensional solution NMR spectroscopy. We found that AtPDFL2.1 displays a strong inhibitory effect against Fusarium graminearum (IC50≈4μM). This peptide folds in the canonical cysteine-stabilized αβ (CSαβ) motif, consisting of one α-helix and one triple-stranded antiparallel β-sheet stabilized by three disulfide bridges and a hydrophobic cluster of residues within its core where the α-helix packs tightly against the β-sheets. Nuclear spin relaxation measurements show that the structure of AtPDFL2.1 is essentially rigid, with the L3 loop located between β-strands 2 and 3 being more flexible and displaying conformational exchange. Interestingly, the dynamic features of loop L3 are conserved among defensins and are probably correlated to the antifungal and receptor binding activities.

Atomic Level Insights into the Activation Mechanism of Neurotensin Receptor 1

Neurotensin receptor type 1 (NTSR1) is a G-protein-coupled Receptor (GPCR) that binds the neuropeptide, neurotensin and regulates vital functions such as cognition, blood pressure, glucose level and digestion. Drugs targeting NTSR1 have the potential for treating addiction, anxiety and loss of memory. Therefore, it is crucial to understand the activation mechanism of this receptor for drug design. The detailed atomic level mechanism of NTSR1 activation is not clearly understood. Previously, deactivation simulations (simulating the transition of the receptor to the inactive state starting from the active conformation) were used to provide insights into the activation mechanism of GPCRs. Here, starting from the active like crystal structure of NTSR1, we have performed several microseconds of MD simulations on the wild type and a constitutively active mutant NTSR1, in an explicit lipid environment both in presence and absence of the agonist neurotensin. In multiple independent simulations, transmembrane helix 7 transitioned into an inactive like conformation within a few hundred nanoseconds, whereas helix 6 continued to be in the active like conformation for several microseconds before transitioning to the inactive conformation. During the deactivation event, a cluster of hydrophobic residues in the transmembrane domain of the receptors showed concerted sidechain movement that facilitated deactivation. One of these residues, L310(6.37) when experimentally mutated to alanine impaired the activation of NTSR1. Both the apoprotein and the neurotensin bound NTSR1 followed similar pathway towards deactivation, although the apoprotein was more flexible compared to the neurotensin bound receptor. Compared to the wild type NTSR1, the constitutively active mutant F358A showed a lower propensity for deactivation. Our observations provide critical insights into the activation of NTSR1 and by inference can be applied to other peptide activated GPCRs.

Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions

CLC-K chloride channels are expressed in the kidney and in the inner ear and require the accessory subunit barttin for proper function and membrane insertion. Barttin exerts multiple functions on CLC-proteins: it modifies protein stability and intracellular trafficking as well as channel activity, ion conduction, and gating. So far, the molecular determinants of these distinct barttin functions have remained elusive. Here we performed serial perturbation mutagenesis to identify the sequence determinants of barttin function. Barttin consists of two transmembrane helices followed by a long intracellular carboxyl terminus, and earlier work demonstrated that the transmembrane core of barttin suffices for most effects on the α-subunit. We individually substituted every amino acid of the predicted transmembrane core (amino acids 9-26 and 35-55) with tryptophan, co-expressed mutant barttin with hClC-Ka or V166E rClC-K1, and characterized CLC-K/barttin channels by patch clamp techniques, biochemistry, and confocal microscopy. The majority of mutations left the chaperone function of barttin, i.e. the effects on endoplasmic reticulum exit and surface membrane insertion, unaffected. In contrast, tryptophan insertion at multiple positions resulted in impaired activity of hClC-Ka/barttin and changes in gating of V166E rClC-K1/barttin. These results demonstrate that mutations in a cluster of hydrophobic residues within transmembrane domain 1 affect barttin-CLC-K interaction and impair gating modification by the accessory subunit. Whereas tight interaction is necessary for functional modification, even impaired association of barttin and CLC-K suffices for normal intracellular trafficking. Our findings allow definition of a likely interaction surface and clarify the mechanisms underlying CLC-K channel modification by barttin.

Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions

Antibodies commonly contain hydrophobic residues within their complementarity-determining regions (CDRs) that mediate binding to target antigens. Unfortunately, hydrophobic CDRs can also promote antibody aggregation, which is especially concerning for therapeutic antibodies due to the immunogenicity of antibody aggregates. Here we investigate how the sequences of CDRs within single-domain (V(H)) antibodies specific for the Alzheimer's amyloid β peptide can be engineered to resist aggregation without reducing binding affinity. We find that domain antibodies containing clusters of hydrophobic residues within their third CDR (CDR3) are prone to aggregate within days at 25°C and minutes above 70°C. However, inserting two or more negatively charged residues at each edge of CDR3 potently suppresses antibody aggregation without altering binding affinity. We also find that inserting charged mutations at one edge of CDR3 (N- or C-terminal) prevents aggregation, but only if such mutations are located at the edge closest to most hydrophobic portion of CDR3. In contrast, charged mutations outside of CDR3 fail to suppress aggregation. Our findings demonstrate that the sequence of CDR loops can be engineered in a systematic manner to improve antibody solubility without altering binding affinity or specificity.

Structure and Function of APH(4)-Ia, a Hygromycin B Resistance Enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.

Solution Structure of the Heterotrimeric Complex between the Interaction Domains of RFX5 and RFXAP from the RFX Gene Regulatory Complex

The mammalian immune response is mediated by a heterotetrameric transcriptional control complex, called regulatory factor X (RFX), that regulates the expression of major histocompatibility complex class II genes. RFX comprises three proteins: RFX5 (two copies), RFXAP, and RFXB, and mutations and deletions that prevent the assembly of the RFX complex have been linked to a severe immunodeficiency disorder. Two RFX5 molecules and one RFXAP molecule assemble in the cytoplasm prior to nuclear localization, a process mediated by an N-terminal “dimerization domain” of RFX5 (RFX5N) and a C-terminal domain of RFXAP (RFXAPC). We previously presented evidence that RFXAPC is unstructured in the absence of RFX5N but adopts a regular structure in the RFX5N2–RFXAPC complex and that the RFX5N2–RFXAPC complex binds RFXB with high affinity. We now report the structure of the RFX5N2–RFXAPC complex, determined in solution by 15N- and 13C-edited NMR spectroscopy. RFX5N consists of a long central helix flanked by two shorter helices. The central helices of the two RFX5N molecules form an antiparallel coiled coil, and the flanking helices pack at the ends of the long helices in a perpendicular arrangement such that the RFX5N dimer is shaped like a staple. RFXAPC consists of two α-helices that form a V-shaped structure that packs within the RFX5N2 staple. Leucine residues in the leucine-rich region of RFX5N (62-LYLYLQL-68) that are critical for major histocompatibility complex class II gene expression in vivo contribute to both the dimer (Leu64 and Leu68) and the RFX5N–RFXAPC interfaces (Leu62 and Leu66). The clustering of hydrophobic residues from different regions of RFXAPC suggests a potential binding site for RFXB.

Crystal Structure of a Bony Fish β2-Microglobulin: INSIGHTS INTO THE EVOLUTIONARY ORIGIN OF IMMUNOGLOBULIN SUPERFAMILY CONSTANT MOLECULES

Crystal Structure of a Bony Fish β2-Microglobulin: INSIGHTS INTO THE EVOLUTIONARY ORIGIN OF IMMUNOGLOBULIN SUPERFAMILY CONSTANT MOLECULES

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

A Patch of Positively Charged Amino Acids Surrounding the Human Immunodeficiency Virus Type 1 Vif SLVx4Yx9Y Motif Influences Its Interaction with APOBEC3G

The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K(22)x(3)K(26)x(3)Y(30)x(9)YRHHY(44)) and a cluster of hydrophobic residues (V(55)xIPLx(4-5)LxPhix2YWxL(72)) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.

Loop-Tryptophan Human Purine Nucleoside Phosphorylase Reveals Submillisecond Protein Dynamics

Human PNP is a homotrimer containing three tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The catalytic sites of PNP are located near the subunit-subunit interfaces where F159 is a catalytic site residue donated from an adjacent subunit. F159 covers the top (beta) surface of the ribosyl group at the catalytic site. QM/MM calculations of human PNP have shown that F159 is the center of the most mobile region of the protein providing access to the substrate in the active site. F159 is also the key residue in a cluster of hydrophobic residues that shield catalytic site ligands from bulk solvent. Trp-free human PNP (Leuko-PNP) was previously engineered by replacing the three Trp residues of native PNP with Tyr. From this active construct, a single Trp residue was placed in the catalytic site loop (F159W-Leuko-PNP) as a reporter group for the ribosyl region of the catalytic site. The F159W-Leuko-PNP fluorescence is red shifted compared to native PNP, suggesting a solvent-exposed Trp residue. Upon ligand binding (hypoxanthine), the 3-fold fluorescence quench confirms conformational packing of the catalytic site pocket hydrophobic cluster. F159W-Leuko-PNP has an on-enzyme thermodynamic equilibrium constant (Keq) near unity in the temperature range between 20 and 30 degrees C and nonzero enthalpic components, making it suitable for laser-induced T-jump analyses. T-jump relaxation kinetics of F159W-Leuko-PNP in equilibrium with substrates and/or products indicate the conformational equilibria of at least two ternary complex intermediates in the nano- to millisecond time scale (1000-10000 s-1) that equilibrate prior to the slower chemical step (approximately 200 s-1). F159W-Leuko-PNP provides a novel protein platform to investigate the protein conformational dynamics occurring prior to transition state formation.

Crystal Structure of Butyrate Kinase 2 from Thermotoga maritima , a Member of the ASKHA Superfamily of Phosphotransferases

The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-A crystal structure of Buk2 complexed with (beta,gamma-methylene) adenosine 5'-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

Synchronous vs Asynchronous Chain Motion in α-Synuclein Contact Dynamics

alpha-Synuclein (alpha-syn) is an intrinsically unstructured 140-residue neuronal protein of uncertain function that is implicated in the etiology of Parkinson's disease. Tertiary contact formation rate constants in alpha-syn, determined from diffusion-limited electron-transfer kinetics measurements, are poorly approximated by simple random polymer theory. One source of the discrepancy between theory and experiment may be that interior-loop formation rates are not well approximated by end-to-end contact dynamics models. We have addressed this issue with Monte Carlo simulations to model asynchronous and synchronous motion of contacting sites in a random polymer. These simulations suggest that a dynamical drag effect may slow interior-loop formation rates by about a factor of 2 in comparison to end-to-end loops of comparable size. The additional deviations from random coil behavior in alpha-syn likely arise from clustering of hydrophobic residues in the disordered polypeptide.