APBSmem: a graphical interface for electrostatic calculations at the membrane

Callenberg KM, Choudhary OP, de Forest GL, Gohara DW, Baker NA, Grabe M

PLoS ONE 2010;5(9)

PMID: 20949122


Electrostatic forces are one of the primary determinants of molecular interactions. They help guide the folding of proteins, increase the binding of one protein to another and facilitate protein-DNA and protein-ligand binding. A popular method for computing the electrostatic properties of biological systems is to numerically solve the Poisson-Boltzmann (PB) equation, and there are several easy-to-use software packages available that solve the PB equation for soluble proteins. Here we present a freely available program, called APBSmem, for carrying out these calculations in the presence of a membrane. The Adaptive Poisson-Boltzmann Solver (APBS) is used as a back-end for solving the PB equation, and a Java-based graphical user interface (GUI) coordinates a set of routines that introduce the influence of the membrane, determine its placement relative to the protein, and set the membrane potential. The software Jmol is embedded in the GUI to visualize the protein inserted in the membrane before the calculation and the electrostatic potential after completing the computation. We expect that the ease with which the GUI allows one to carry out these calculations will make this software a useful resource for experimenters and computational researchers alike. Three examples of membrane protein electrostatic calculations are carried out to illustrate how to use APBSmem and to highlight the different quantities of interest that can be calculated.

Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase

Marcotte LL, Wass AB, Gohara DW, Pathak HB, Arnold JJ, Filman DJ, Cameron CE, Hogle JM

J. Virol. 2007 Apr;81(7):3583-96

PMID: 17251299


Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C(pro) and the viral polymerase 3D(pol) and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-A resolution and the G64S fidelity mutant of 3D(pol) at a 3.0-A resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D(pol) G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain’s N terminus to the VPg binding site suggest how 3D(pol) makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

Poliovirus RNA-dependent RNA polymerase (3Dpol): kinetic, thermodynamic, and structural analysis of ribonucleotide selection

Gohara DW, Arnold JJ, Cameron CE

Biochemistry 2004 May;43(18):5149-58

PMID: 15122880


We have performed a kinetic and thermodynamic analysis of 3D(pol) derivatives containing substitutions in the ribose-binding pocket with ATP analogues containing correct and incorrect sugar configurations. We find that Asp-238, a residue in structural motif A that is conserved in all RNA-dependent RNA polymerases, is a key determinant of polymerase fidelity. Alterations in the position of the Asp-238 side chain destabilize the catalytically competent 3D(pol)-primer/template-NTP complex and reduce the efficiency of phosphoryl transfer. The reduction in phosphoryl transfer may be a reflection of increased mobility of other residues in motif A that are required for stabilizing the triphosphate moiety of the nucleotide substrate in the active conformation. We present a structural model to explain how Asp-238 functions to select nucleotides with a correct sugar configuration and a correct base. We propose that this mechanism is employed by all RNA-dependent RNA polymerases. We discuss the possibility that all nucleic acid polymerases with the canonical “palm”-based active site employ a similar mechanism to maximize fidelity.

Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+

Arnold JJ, Gohara DW, Cameron CE

Biochemistry 2004 May;43(18):5138-48

PMID: 15122879


The use of Mn(2+) as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg(2+) often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D(pol)) in the presence of Mn(2+). The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg(2+) and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn(2+), the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn(2+), it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg(2+), highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn(2+) to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg(2+), this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn(2+) may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg(2+).

Manganese-dependent polioviruses caused by mutations within the viral polymerase

Crotty S, Gohara D, Gilligan DK, Karelsky S, Cameron CE, Andino R

J. Virol. 2003 May;77(9):5378-88

PMID: 12692240


Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3D(pol), we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn(2+)-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.

Structure-function relationships of the RNA-dependent RNA polymerase from poliovirus (3Dpol). A surface of the primary oligomerization domain functions in capsid precursor processing and VPg uridylylation

Pathak HB, Ghosh SK, Roberts AW, Sharma SD, Yoder JD, Arnold JJ, Gohara DW, Barton DJ, Paul AV, Cameron CE

J. Biol. Chem. 2002 Aug;277(35):31551-62

PMID: 12077141


The primary oligomerization domain of poliovirus polymerase, 3Dpol, is stabilized by the interaction of the back of the thumb subdomain of one molecule with the back of the palm subdomain of a second molecule, thus permitting the head-to-tail assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm is key to the stability of this interface. We show that mutations predicted to completely disrupt this interface do not produce equivalent growth phenotypes. Virus encoding a polymerase with changes of both residues of the thumb to alanine is not viable; however, virus encoding a polymerase with changes of all three residues of the palm to alanine is viable. Biochemical analysis of 3Dpol derivatives containing the thumb or palm substitutions revealed that these derivatives are both incapable of forming long fibers, suggesting that polymerase fibers are not essential for virus viability. The RNA binding activity, polymerase activity, and thermal stability of these derivatives were equivalent to that of the wild-type enzyme. The two significant differences observed for the thumb mutant were a modest reduction in the ability of the altered 3CD proteinase to process the VP0/VP3 capsid precursor and a substantial reduction in the ability of the altered 3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this reaction. Because 3C alone can substitute for 3CD in this reaction, we conclude that the lethal replication phenotype associated with the thumb mutant is caused, in part, by the disruption of an interaction between the back of the thumb of 3Dpol and some undefined domain of 3C. We speculate that this interaction may also be critical for assembly of other complexes required for poliovirus genome replication.

Poliovirus RNA-dependent RNA polymerase (3Dpol): structural, biochemical, and biological analysis of conserved structural motifs A and B

Gohara DW, Crotty S, Arnold JJ, Yoder JD, Andino R, Cameron CE

J. Biol. Chem. 2000 Aug;275(33):25523-32

PMID: 10827187


We have constructed a structural model for poliovirus RNA-dependent RNA polymerase (3D(pol)) in complex with a primer-template (sym/sub) and ATP. Residues found in conserved structural motifs A (Asp-238) and B (Asn-297) are involved in nucleotide selection. Asp-238 appears to couple binding of nucleotides with the correct sugar configuration to catalytic efficiency at the active site of the enzyme. Asn-297 is involved in selection of ribonucleoside triphosphates over 2′-dNTPs, a role mediated most likely via a hydrogen bond between the side chain of this residue and the 2′-OH of the ribonucleoside triphosphate. Substitutions at position 238 or 297 of 3D(pol) produced derivatives exhibiting a range of catalytic efficiencies when assayed in vitro for poly(rU) polymerase activity or sym/sub elongation activity. A direct correlation existed between activity on sym/sub and biological phenotypes; a 2.5-fold reduction in polymerase elongation rate produced virus with a temperature-sensitive growth phenotype. These data permit us to propose a detailed, structural model for nucleotide selection by 3D(pol), confirm the biological relevance of the sym/sub system, and provide additional evidence for kinetic coupling between RNA synthesis and subsequent steps in the virus life cycle.

Production of “authentic” poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli

Gohara DW, Ha CS, Kumar S, Ghosh B, Arnold JJ, Wisniewski TJ, Cameron CE

Protein Expr. Purif. 1999 Oct;17(1):128-38

PMID: 10497078


The first amino acid of “authentic” poliovirus RNA-dependent RNA polymerase, 3D(pol), is a glycine. As a result, production of 3D(pol) in Escherichia coli requires addition of an initiation codon; thus, a formylmethionine is added to the amino terminus. The formylmethionine should be removed by the combined action of a cellular deformylase and methionine aminopeptidase. However, high-level expression of 3D(pol) in E. coli yields enzyme with a heterogeneous amino terminus. To preclude this problem, we developed a new expression system for 3D(pol). This system exploits the observation that proteins fused to the carboxyl terminus of ubiquitin can be processed in E. coli to produce proteins with any amino acid as the first residue when expressed in the presence of a ubiquitin-specific, carboxy-terminal protease. By using this system, authentic 3D(pol) can be obtained in yields of 30-60 mg per liter of culture. While addition of a single glycine, alanine, serine, or valine to the amino terminus of 3D(pol) produced derivatives with a specific activity reduced by at least 25-fold relative to wild-type enzyme, addition of a methionine to the amino terminus resulted in some processing to yield enzyme with a glycine amino terminus. Addition of a hexahistidine tag to the carboxyl terminus of 3D(pol) had no deleterious effect on the activity of the enzyme. The utility of this expression system for production of other viral polymerases and accessory proteins is discussed.