Amino Acid Residues For Binding Research Articles

Interaction interface of human flap endonuclease-1 with its DNA substrates.

Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. To map further the corresponding interactions between FEN-1 residues and DNA substrates, we performed biochemical assays employing a series of flap DNA substrates lacking some structural components and a series of binding-deficient point mutants of FEN-1. It was revealed that Arg(47), Arg(70), and Lys(326)-Arg(327) of FEN-1 interact with the upstream duplex of DNA substrates, whereas Lys(244)-Arg(245) interact with the downstream duplex. This result indicates the orientation of the FEN-1-DNA interaction. Moreover, Arg(70) and Arg(47) were determined to interact with the sites around the 2nd nucleotide (Arg(70)) or the 5th/6th nucleotide (Arg(47)) of the template strand in the upstream duplex portion counting from the nick point of the flap substrate. Together with previously published data and the crystallographic ainformation from the FEN-1.DNA complex that we published recently (Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J. A. (2004) Cell 116, 39-50) we are able to propose a reasonable model for how the human FEN-1 protein interacts with its DNA substrates.

Identification of five new HLA-B*3501-restricted epitopes derived from common melanoma-associated antigens, spontaneously recognized by tumor-infiltrating lymphocytes.

We previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed melanoma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL- and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients.

Sequence-specific Binding of prePhoD to Soluble TatAd Indicates Protein-mediated Targeting of the Tat Export in Bacillus subtilis

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.

Inhibition of complement activation by recombinant Sh-CRIT-ed1 analogues.

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.

Region and amino acid residues required for Rad51C binding in the human Xrcc3 protein.

The Xrcc3 protein, which is required for the homologous recombinational repair of damaged DNA, forms a complex with the Rad51C protein in human cells. Mutations in either the Xrcc3 or Rad51C gene cause extreme sensitivity to DNA-damaging agents and generate the genomic instability frequently found in tumors. In the present study, we found that the Xrcc3 segment containing amino acid residues 63-346, Xrcc3(63-346), is the Rad51C-binding region. Biochemical analyses revealed that Xrcc3(63-346) forms a complex with Rad51C, and the Xrcc3(63-346)- Rad51C complex possesses ssDNA and dsDNA binding abilities comparable to those of the full-length Xrcc3-Rad51C complex. Based on the structure of RecA, which is thought to be the ancestor of Xrcc3, six Xrcc3 point mutants were designed. Two-hybrid and biochemical analyses of the Xrcc3 point mutants revealed that Tyr139 and Phe249 are essential amino acid residues for Rad51C binding. Superposition of the Xrcc3 Tyr139 and Phe249 residues on the RecA structure suggested that Tyr139 may function to ensure proper folding and Phe249 may be important to constitute the Rad51C-binding interface in Xrcc3.

NO運搬タンパク質NP4の外来性配位子結合に対するアミノ酸残基の役割

NO運搬タンパク質NP4の外来性配位子結合に対するアミノ酸残基の役割

Mutational analysis of bacteriophage T4 AsiA: involvement of N- and C-terminal regions in binding to σ 70 of Escherichia coli in vivo

The T4 AsiA is an anti-sigma factor encoded by an early gene of bacteriophage T4. AsiA has been shown to inhibit T4 early promoters in vitro and expression of this protein from a plasmid causes transcriptional shut off in the host cells leading to cell death. By reasoning that mutant AsiA expression in Escherichia coli will not inhibit the host transcription and hence lead to healthy colony formation, a strategy was developed wherein inactive or partially active mutants of AsiA could be isolated. These mutants were tested for their ability to bind to σ 70 in vivo in E. coli, monitored as a relative toxicity assay, co-purification of σ 70, inhibition of [ 3H-uridine] incorporation and also in the yeast two hybrid system. A good correlation was found between the loss of toxicity of AsiA to E. coli cells and the inability of mutant AsiAs to bind to σ 70 It was observed that deletion of C-terminal 17 amino acid residues of AsiA did not affect the activity whereas a mutant asiA lacking C-terminal 28 amino acid residues had the toxicity reduced to a large extent, suggesting that amino acid residues between 64 and 73 played a role in binding to AsiA. A mutant with a deletion of 34 amino acids in the C-terminus did not show any toxicity to E. coli cells. In the N-terminal region, deletion of five amino acid residues was tolerated but extending the deletion to ten amino acids abolished the AsiA activity completely. The conversion of glutamic acid (E10) to either leucine, serine, glutamine, tyrosine or alanine did not affect the toxicity to a great extent suggesting that a negative charge at E10 is not critical for interaction with σ 70. The results of our in vivo studies suggest that the primary σ 70 binding site of AsiA is in N-terminus, but, it requires the presence of C-terminal 64–73 amino acid residues for effective binding in vivo.

Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system.

Cytochrome P450foxy (P450foxy, CYP505) is a fused protein of cytochrome P450 (P450) and its reductase isolated from the fungus Fusarium oxysporum, which catalyzes the subterminal (omega-1 approximately omega-3) hydroxylation of fatty acids. Here, we produced, purified and characterized a fused recombinant protein (rP450foxy) using the Escherichia coli expression system. Purified rP450foxy was catalytically and spectrally indistinguishable from the native protein, but most of the rP450foxy was recovered in the soluble fraction of E. coli cells unlike the membrane-bound native protein. The results are consistent with our notion that the native protein is targeted to the membrane by a post-translational modification mechanism. We also discovered that P450foxy could use shorter saturated fatty acid chains (C9 and C10) as a substrate. The regiospecificity (omega-1 approximately omega-3) of hydroxylation due to the enzymatic reaction for the short substrates (decanoate, C10; undecanoate, C11) was the same as that for longer substrates. Steady state kinetic studies showed that the kcat values for all substrates tested (C9-C16) were of the same magnitude (1200-1800 min-1), whereas the catalytic efficiency (kcat/Km) was higher for longer fatty acids. Substrate inhibition was observed with fatty acid substrates longer than C13, and the degree of inhibition increased with increasing chain length. This substrate inhibition was not apparent with P450BM3, a bacterial counterpart of P450foxy, which was the first obvious difference in their catalytic properties to be identified. Kinetic data were consistent with the inhibition due to binding of the second substrate. We discuss the inhibition mechanism based on differences between P450foxy and P450BM3 in key amino acid residues for substrate binding.

Cytochrome aa(3) in Haloferax volcanii.

A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN(-) complex spectrum that indicates the presence of heme a and heme a(3). This cytochrome aa(3) consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa(3), providing physiological evidence for electron transfer from cytochrome c to cytochrome aa(3) in archaea.

Structure-function analysis of photosystem II subunit S (PsbS) in vivo.

In land plants, photosystem II subunit S (PsbS) plays a key role in xanthophyll- and pH-dependent non-photochemical quenching (qE) of excess absorbed light energy. Arabidopsis thaliana (L.) Heynh. npq4 mutants are defective in the psbS gene and have impaired qE. Exactly how the PsbS protein is involved in qE is unclear, but it has been proposed that PsbS binds H+ and/or de-epoxidized xanthophylls in excess light as part of the qE mechanism. To identify amino acid residues that are important for PsbS function, we sequenced the psbS gene from eight npq4 point mutant alleles isolated by forward genetics screening, including two new alleles. In the four transmembrane helices of PsbS, several amino acid residues were found to affect the stability and/or function of the protein. By comparing the predicted amino acid sequences of PsbS from several plant species and studying the proposed topological structure of PsbS, eight possible H+-binding amino acid residues on the lumenal side of the protein were identified and then altered by site-directed mutagenesis in vitro. The mutant psbS genes were transformed into npq4-1, a psbS deletion mutant, to test the stability and function of the mutant PsbS proteins invivo. The results demonstrate that two conserved, protonatable amino acids, E122 and E226, are especially critical for the function of PsbS.

Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase.

Binding of 14-3-3 proteins to nitrate reductase phosphorylated on Ser543 (phospho-NR) inhibits activity and is responsible for the inactivation of nitrate reduction that occurs in darkened leaves. The 14-3-3-dependent inactivation of phospho-NR is known to require millimolar concentrations of a divalent cation such as Mg2+ at pH 7.5. We now report that micromolar concentrations of the polyamines, spermidine(4+) and spermine(3+), can substitute for divalent cations in modulating 14-3-3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine(4+) > spermidine(3+) >>> cadavarine(2+) approximately putrescine(2+) approximately agmatine(2+) approximately N1-acetylspermidine(2+), indicating that two primary and at least one secondary amine group were required. C-terminal truncations of GF14 omega, which encodes the Arabidopsis 14-3-3 isoform omega, indicated that loop 8 (residues 208-219) is the likely cation-binding site. Directed mutagenesis of loop 8, which contains the EF hand-like region identified in earlier studies, was performed to test the role of specific amino acid residues in cation binding. The E208A mutant resulted in a largely divalent cation-independent inhibition of phospho-NR activity, whereas the D219A mutant was fully Mg(2+)-dependent but had decreased affinity for the cation. Mutations and C-terminal truncations that affected the Mg(2+) dependence of phospho-NR inactivation had similar effects on polyamine dependence. The results implicate loop 8 as the site of divalent cation and polyamine binding, and suggest that activation of 14-3-3s occurs, at least in part, by neutralization of negative charges associated with acidic residues in the loop. We propose that binding of polyamines to 14-3-3s could be involved in their regulation of plant growth and development.

Structure of E. coli 5′-methylthioadenosine/S-adenosylhomocysteine Nucleosidase Reveals Similarity to the Purine Nucleoside Phosphorylases

Background: 5′-methylthioadenosine/S-adenosyl-homocysteine (MTA/AdoHcy) nucleosidase catalyzes the irreversible cleavage of 5′-methylthioadenosine and S-adenosylhomocysteine to adenine and the corresponding thioribose, 5′-methylthioribose and S-ribosylhomocysteine, respectively. While this enzyme is crucial for the metabolism of AdoHcy and MTA nucleosides in many prokaryotic and lower eukaryotic organisms, it is absent in mammalian cells. This metabolic difference represents an exploitable target for rational drug design. Results: The crystal structure of E. coli MTA/AdoHcy nucleosidase was determined at 1.90 Å resolution with the multiwavelength anomalous diffraction (MAD) technique. Each monomer of the MTA/AdoHcy nucleosidase dimer consists of a mixed α/β domain with a nine-stranded mixed β sheet, flanked by six α helices and a small 3 10 helix. Intersubunit contacts between the two monomers present in the asymmetric unit are mediated primarily by helix-helix and helix-loop hydrophobic interactions. The unexpected presence of an adenine molecule in the active site of the enzyme has allowed the identification of both substrate binding and potential catalytic amino acid residues. Conclusions: Although the sequence of E. coli MTA/AdoHcy nucleosidase has almost no identity with any known enzyme, its tertiary structure is similar to both the mammalian (trimeric) and prokaryotic (hexameric) purine nucleoside phosphorylases. The structure provides evidence that this protein is functional as a dimer and that the dual specificity for MTA and AdoHcy results from the truncation of a helix. The structure of MTA/AdoHcy nucleosidase is the first structure of a prokaryotic nucleoside N-ribohydrolase specific for 6-aminopurines.

Archaeoglobus fulgidus RNase HII in DNA Replication: Enzymological Functions and Activity Regulation via Metal Cofactors

RNA primer removal during DNA replication is dependent on ribonucleotide- and structure-specific RNase H and FEN-1 nuclease activities. A specific RNase H involved in this reaction has long been sought. RNase HII is the only open reading frame in Archaeoglobus fulgidus genome, while multiple RNases H exist in eukaryotic cells. Data presented here show that RNase HII from A. fulgidus (aRNase HII) specifically recognizes RNA–DNA junctions and generates products suited for the FEN-1 nuclease, indicating its role in DNA replication. Biochemical characterization of aRNase HII activity in the presence of various divalent metal ions reveals a broad metal tolerance with a preference for Mg2+ and Mn2+. Combined mutagenesis, biochemical competitions, and metal-dependent activity assays further clarify the functions of the identified amino acid residues in substrate binding or catalysis, respectively. These experiments also reveal that Asp129 form a second-metal binding site, and thus contribute to activity attenuation.

Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor: N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY

Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor: N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY

Calculation of Hydration Effects in the Binding of Anionic Ligands to Basic Proteins

The accurate calculation of the energetics of electrostatically driven binding of amino acid residues to other amino acids (salt bridges) or to synthetic molecules is critical in numerous physiological and technological processes. Commonly used continuum methods are unable to capture differences in interaction energies resulting from specific chemical changes, such as the stronger retention of basic proteins on sulfated (strong) than on carboxylated (weak) cation exchangers. The inadequacies of continuum models in describing hydration effects, specifically local solvent structure and associated polarization effects, are especially apparent in such systems. These shortcomings have been addressed by modeling the protein−cation exchanger interaction as that of methylammonium, a model for a protonated lysine residue, with the methylated analogues of the ion-exchange functionalities, namely methyl sulfate and acetate ion, respectively. A hybrid quantum-continuum treatment of the solvent is able to capture the qualitative differences in binding that are not obtained by continuum methods. More remarkably, it is seen that a heuristic approach, based solely on the bulk solvation free energies of individual species, is able to describe qualitatively the differences in binding.

A Dynamic Model for Bilirubin Binding to Human Serum Albumin

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.

Platelet glycoprotein IIb/IIIa receptors and Glanzmann's thrombasthenia.

Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, a system designed to act quickly and effectively to arrest hemorrhage. This system is also triggered by pathogenic events, such as the rupture of an atherosclerotic plaque, which can lead to thrombotic vaso-occlusion, ischemia, and infarction. Platelets are a major contributor to these damaging and life-threatening thrombotic phenomena because of their adhesive properties, which result in the release of soluble mediators, platelet aggregation, and enhancement of thrombin generation.1 The platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is a key component in the pathway to platelet aggregation; consequently, this receptor has become the target for therapeutic intervention. A paradigm of this antiplatelet treatment modality is found naturally in the inherited disorder Glanzmann’s thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding but only rarely demonstrate spontaneous central nervous system hemorrhage,2 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann’s thrombasthenia result in a functional deficiency of GPIIb/IIIa receptors,2 3 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. The molecular characterization of mutations causing Glanzmann’s thrombasthenia has provided a wealth of information on structure-function relations of the GPIIb/IIIa receptor. This review will briefly summarize those mutations that affect ligand-binding domains and receptor activation and present them in the context of predicted structures. More comprehensive coverage can be found in reviews discussing the structure and function of the GPIIb/IIIa receptor complex4 5 and the clinical and molecular basis of Glanzmann’s thrombasthenia.2 3 Platelets are the first line of defense in preventing blood loss from injured blood vessels via recognition and adhesion to components of the subendothelial matrix. This event is followed by formation of a platelet …

Identification of specific histidine residues and the carboxyl terminus are essential for serotonin N-acetyltransferase enzymatic activity

Melatonin is synthesized in pinealocytes of the pineal gland and in photoreceptors of the retina. Synthesis rate from serotonin to melatonin is controlled by the rapid and dramatic enzymatic increase in darkness of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, EC 2.3.1.87) and hydroxyindole- O-methyltransferase (HIOMT, EC 2.1.1.4). The primary structure of these critical indoleamine enzymes is now known and the regulation of the enzyme catalysis can be examined. As a first step, the conserved cysteine (C) and histidine (H) residues were targeted for site-directed mutagenesis as potential amino acid residues involved in the N-acetylation reaction of AA-NAT. Our studies concluded that among 6 histidine (H) to alanine (A) mutations, three residues (H110A, H118A, H120A) within the AA-NAT protein showed little or no enzymatic activity, whereas the others (H28A, H70A, H125A) retained enzymatic activity, compared to the unaltered AA-NAT protein. Cysteine to alanine mutations, C37A and C177A, had no significant effect on the AA-NAT enzymatic activity; however, C61A had a four-fold increase in K m for acetyl CoA and an altered sensitivity to the thiol modification chemical, N-ethylmaleimide (NEM), implying that C61 may participate in the acetyl CoA binding. Further studies examined the AA-NAT enzyme regulation of the highly conserved carboxyl terminus. When 12 terminal amino acid residues were deleted systematically from the carboxyl terminus of the 205 amino acid residue AA-NAT protein, enzyme activity was retained. However, further residue deletion resulted in enzyme activity plummeting, implicating that the essential information either for the correct structural folding into an active enzyme form or for enzyme stability is in the 193 residues. To test the relative importance of the AA-NAT carboxyl terminal region, a single leucine (L) was altered to alanine (A) or proline (P). Both mutants, either L193A or L193P, had a marked decrease in AA-NAT enzymatic activity and a decrease in thermal stability, suggesting the leucine, in addition to the cysteine and histidine residues, is involved in either enzyme catalysis or stability. In light of the recently reported three-dimensional structure of AA-NAT (17,18), the site-directed mutagenesis data demonstrate experimentally the importance of essential amino acid residues for acetyl CoA binding and AA-NAT activation.

Identification by site-directed mutagenesis of amino acid residues in ribosomal protein L2 that are essential for binding to 23S ribosomal RNA

The ribosomal protein L2 ( BstL2) from Bacillus stearothermophilus is a primary 23S rRNA binding protein. We made use of site-directed mutagenesis to identify essential basic and aromatic amino acid residues for 23S rRNA binding. Four mutants, R68Q, K70Q, R86Q, and R155Q, in which Arg-68, Lys-70, Arg-86, and Arg-155, respectively, are replaced by the Gln residue, showed reduced binding affinities as compared with that of the wild type BstL2 (a binding constant K=8.93 μM −1): K values of these mutants range between 0.24 and 1.86 μM −1. As for aromatic amino acids, replacements of Phe-66, Tyr-95 or Tyr-102 by alanine significantly abolished the binding affinities. CD analysis of the mutant proteins indicated that the mutations of four basic residues (Arg-68, Lys-70, Arg-86 and Arg-155) did not affect protein structure, whereas those of aromatic residues (Phe-66, Tyr-95, and Tyr-102) appeared to cause slight structural perturbations. These results, together with sequence comparison of L2 family proteins, suggest that Arg-86 and Arg-155 in BstL2 may act as positively charged recognition groups for negatively charged phosphate backbone of the 23S rRNA, and that Phe-66, Tyr-95, and Tyr-102 may be candidate residues which stabilize the BstL2-23S rRNA interaction through intramolecular interactions.

Anatomy of hot spots in protein interfaces

Binding of one protein to another is involved in nearly all biological functions, yet the principles governing the interaction of proteins are not fully understood. To analyze the contributions of individual amino acid residues in protein-protein binding we have compiled a database of 2325 alanine mutants for which the change in free energy of binding upon mutation to alanine has been measured (available at http://motorhead.ucsf.edu/∼thorn/hotspot). Our analysis shows that at the level of side-chains there is little correlation between buried surface area and free energy of binding. We find that the free energy of binding is not evenly distributed across interfaces; instead, there are hot spots of binding energy made up of a small subset of residues in the dimer interface. These hot spots are enriched in tryptophan, tyrosine and arginine, and are surrounded by energetically less important residues that most likely serve to occlude bulk solvent from the hot spot. Occlusion of solvent is found to be a necessary condition for highly energetic interactions.